STUDIES ON HISTOPATHOLOGICAL TRANSFORMATIONS INDUCED BY MELOIDOGYNE INCOGNITA IN BITTERGOURD, MOMORDICA CHARANTIA
ABSTRACT
THESIS SUBMITTED FOR THE DEGREE OF Doctor of Philosophy IN BOTANY
BY MOHD. YAQUB BHAT T'S^Sl
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 1999 I Acc. N- )*'
'"'hat Univc^ ABSTRACT
Momordica charantia, an important vegetable, belonging to the family Cucurbitaceae, is grown during summer throughout India. Often, the roots of plants are attacked by the root -knot nematode, Meloidogyne incognita that causes stunting, wilting, and yellowing of above ground parts and galling on the roots. Deformations in the galled roots affect absorption of water and minerals by the root and transport of these substances towards the shoot. The plant, at the initial stages of infection, shows wilting symptoms, the plant growth retards, the leaves become chlorotic, and finally the plant dies. The ultimate impact of the disease is the yield loss that may reach upto fifty percent.
The experiments were carried out in various stages. In one phase the infected plants were compared with uninfected control plants. In the other phase disease controlling measures were applied and their effects on plants and nematode were studied.
The overall growth of infected plants when treated with Paecilomyces lilacinus increased. The plant growth increased to a greater extent when the fungus was applied into the soil one week before nematode inoculation, when compared with nematode inoculated and untreated plants. In Tj plants, the lengths and weights of roots and shoots increased to such an extent that the plant growth became almost equal to { 2 } that of control plants. The fungus was found spreading in the soil, around the root surface, and in the inner tissues of the root. The fungus after penetrating into the root tissues grew inter-and intracellularly. It preferred the lumen of vessel elements, during intracellular development. The hyphae were observed in the giant cells and also in the body of the mature female. It damaged and destroyed eggs and egg masses and ultimaterly checked secondary infection. A decreasing trend in plant growth parameters was observed as the time of application of the fungus increased. In comparison to nematode inoculated and untreated plants, the treated plants exhibited an increase in number of branches, number of flowers, and leaf area. In the vascular tissue, less disruption was observed in the treated plants.
In the second experiment, aldicarb a non-fumigant systemic nematicide was used to study its effect on the development of the nematode and on the plant growth. The nematicide was applied simultaneously at the time of inoculation and at an interval of one week to four weeks after nematode inoculation.
The Tj plants in which nematicide was applied simultaneously alongwith nematode inoculation exhibited an enormous increase in plant growth as compared to nematode inoculated_and untreated olants. In T 3 plants the lengths and weights of roots and shoots, number of branches, leaf area, number of flowers increased. In these plants size of the gall, number of the galls, number of mature females, number of egg masses { 3 } were lower. When the application time increased from one week to four weeks, the growth of the plant decreased, number of galls, size of galls, number of mature females and number of egg masses increased. The anatomical studies of galled roots revealed most disruption in T^ plants and least disruption in T^ plants. The galls of T3 plants contatined less amount of abnormal xylem and abnormal pl^^m as compared to other treatments.
The third experiment was aimed at knowing the effect of different inoculum levels of the nematode on the growth of the plant, on the formation of the galls, on the development of the nematodes, and on the formation of abnormal tissues inside the galls.
An initial population density of Meloidogyne incognita affects different host plants differently. Low population level either may not affect a plant, or it may be beneficial or harmful. Momordica charantia responded differently to different population densities. At the lowest inoculum level there was not any remarkable decline in growth of plant as compared to control. At 50 Jj level, a slight but non-significant increase was observed. At higher initial inoculum levels the growth decreased significantly. Reduction in lengths and weights of both roots and shoots was maximum at 5,000 J„ the highest inoculum level.
The galls were scanty and very small at lowest (Pi=05 Jj) initial inoculum level. The gall number and the gall size increased from lower to higher inoculum levels wi^h the maximum at highest inoculum level. { 4 }
The number of mature females recovered from plants at Pi = 05 J^ was low that increased to maximum at Pi = 5,000 J^. However, their size decreased as the inoculum level increased.
At lowest inoculum level one nematode was enough to cause the formation of giant cell complex. While at higher inoculum levels more nematodes were found causing multiple giant cell complexes. The average size of giant cell was larger at lower Pi and smaller at higher inoculum level. The giant cell cytoplasm was more dense at lower inoculum level than at higher inoculum level. Abnormalities in the orientation and structure of xylem and phloem were few at lower initial inoculum level and more at higher inoculum levels.
At lowest Pi the number of giant cells around the head of the mature female was 6 to 8. The gaint cells were larger in size enclosed dense cytoplasm as compared to those found in the plant at high inoculum level. The amount of abnormal xylem and phloem was more at higher inoculum level than at lower inoculum level. Out of four primary inoculum levels (O5J2, SOJj, SOOJj and 5,000J2) considered, the galling was scanty and also the size of the gall was very small at the lowest inoculum level. The number of mature females recorded was small, at lowest inoculum level, however, their size was large as compared to the other inoculum levels. At this level the number of egg masses obtained was large.
By the increase in primary inoculum level, the plant growth gradually reduced, number and size of the gall increased, number of { 5 } mature females per gram root increased but the size of the mature female decreased. The number of egg masses per plant also increased. Greatest reduction in length and weight of plant was observed at the highest initial inoculum level. The number and the size of the gall, the number of mature females per gram root and the number of egg masses per plant were maximum at the highest inoculum level. The size of the mature female decreased at higher inoculum levels.
In another experiment different varieties of bittergourd available in the market were examined to find out any Meloidogyne incognita resistant variety. This experiment was conducted because previously tested resistant variety is not available. The six varieties viz Faizabadi, Jhalarwali, PDM, Jaunpuri, Baramasi and Aligarh local obtained from different seed sources were evaluated for this purpose.
Out of the six different varieties of Momordica charantia selected, Aligarh local and Baramasi exhibited highest reduction in length and weight at various initial inoculum levels, as compared to control. The number and size of the galls and the number of the mature females were more in these two varieties. The varietv Faizabadi exhibited least effect of Meloidogyne incognita on plant growth. The gall number was lowest in this variety. In Jhalarwali variety the reduction in plant growth was more than Faizabadi but lower than other four varieties. It produced more galls than Faizabadi. The plant growth of nematode infected PDM and Jaunpuri plants was lower than Faizabadi and Jhalarwali but higher ( 6 } than Baramasi and Aligarh Local.
In the variety Faizabadi no galling was observed and the galls if produced were small. In these galls, the giant cells were small, and contained little cytoplasm. The nematode either died or did not reach to mature stage. The galled regions were not distorted. Abnormal xylem and phloem was scarce. In other varieties number and size of galls, size of giant cells increased, being maximum in Aligarh local followed by Baramasi, Jaunpuri, PDM and Jhalarwali.
In the last experiment Meloidogyne incognita infected roots were examined from the day one to the 30th day, after inoculation. At regular intervals of time anatomical studies were carried out to investigate sequential changes in the formation of giant cells, in the development of the nematode, and in the formation of hypertrophic and hyperplastic tissue and abnormal vascular elements.
The characteristic feature in Meloidogyne induced galls is the formation of discrete, abnormally large giant cells. In growing roots, the juveniles of M. incognita induced giant cell in provascular elements, especially from the cells which develop into primary phloem. Soon after penetration, the juveniles entered the procambium zone and incited hypertrophy and hyperplasia not only in sieve tube transforming cells but also in the nearby cells. A large number of cells around the nematode head enlarged and many of them divided leading to the formation of the gall. The affected cells comprised of the cells of { 7 } cortex, endodermis, pericycle, conjuctive tissue, xylem, phloem and pith parenchyma.
The juveniles of Meloidogyne incognita penetrated at or behind the root tips of Momordica charantia. They migrated intercellularly, in the inner tissue, by separating the cell walls. The giant cells were induced in the region of undifferentiated phloem within 48h of inoculation. Maximum number of nuclei and highly dense cytoplasm was noticed after 12 days of inoculation. Decrease in number of nuclei and increase in vacuolation was found after 18 days of inoculation. Smaller giant cells became empty and changed into vessel like elements by the deposition of lignified secondary wall material. Larger giant cells with little or no cytoplasm also transformed into abnormal vessel elements, after 30 days of inoculation.
Secondary infection was noticed after 30 days of inoculation. The egg masses of all the females were not expelled out of the plant tissue. Some egg masses remained inside. Eggs of these egg masses hatched and the second-stage juveniles, traversing intercellularly through cortical cells, reached cambial zone and caused secondary infection. Repetition of hypertrophic and hyperplastic reactions, due to secondary infection, caused a rapid increase in gall size. Some freshly hatched second-stage juveniles were found associated with preformed gaint cells. These juveniles, instead of inducing new giant cells, started feeding on old giant cells. Hypertrophic and hyperplastic reactions were accompanied with such type of feeding. { 8 }
As far as abnormalities in vascular elements are concerned, the abnormality in orientation of vascular strands was started 48h after inoculation.Vascular strands were severely distorted due to the multiple hypertrophic and hyperplastic reactions taking place continuously. The strands became wavy and appeared as scattered patches,when seen in longitudinal sections. The vessel elements became structurally abnormal within 48h of inoculation. The vessel elements broadened near giant cells due to hypertrophic reactions. Irregular shape and size of vessel elements of metaxylem strands are the result of pressure exerted on them by the tissue resulting from hypertrophic and hyperplastic reactions. Abnormal origin of vessel elements was observed 72h after inoculation from small parenchyma cells; 12 days after inoculation from hypertrophied parenchyma cells; 18 days after inoculation from small giant cells; and 30 days after inoculation from larger giant cells. From these observations, it is suggested that formation of abnormal vessel elements in excess amount might increase upward translocation, or supply water to giant cells, or provide protection to giant cells, or gives support to entire gall to prevent its collapse.
From our study it may be concluded that, although giant cells appeared completely enclosed by abiu-omal xylem elements, but the serial section study revealed that none of the giant cell was completely enveloped by the xylem. The giant cells were always connected with the phloem. The sieve tube elements as seen in transverse section, instead of { 9 }
forming a complete ring, appeared diverting towards the giant cells. In this way the supply of assimilates to the giant cells did not disrupt.
The fungus Paecilomyces lilacinus when applied into the soil one week before inoculation of Meloidogyne incognita resulted in : (i) increase in length of roots and shoots, (ii) increase in weight of roots and shoots, (iii) increase in number of branches, (iv) increase in leaf area, (v) increase in number of flowers, / (vi) decrease in gall size, y (vii) decrease in gall number, ^(viii)decrease in number of mature females per gram root, . (ix) decrease in number of egg masses, - (x) decrease in abnormal xylem and abnormal phloem formation, / (xi) destruction of eggs and egg masses, and / (xii) total check of secondary infection.
Later applications diminished the efficacy of the fungus.
The systemic nematicide aldicarb when applied into the soil simultaneously along with the nematode inoculation caused : (i) increase in length of roots and shoots, (ii) increase in weight of roots and shoots, (iii) increase in number of branches, (iv) increase in leaf area, { 10 }
(v) increase in number of flowers, (vi) decrease in gall size, (vii) decrease in gall number, (viii)decrease in number of mature females per gram root, (ix) decrease in number of egg masses, (x) decrease in abnormal xylem and abnormal phloem formation, (xi) death of second stage juveniles of second cycle, and (xii) complete check of secondary infection. Application of aldicarb at later intervals diluted its efficacy.
The initial inoculum levels of Meloidogyne incognita comprising of OSJj, SOJj, SOOJj and 5,000 J^ produced varying effects on the plants. There was no any significant change in plant growth at OSJ^ and SOJ^ inoculum levels. Significant effects were observed at 5,000 J^ inoculum level where :
(i) the length of roots and shoots decreased, (ii) the weight of roots and shoots decreased, (iii) the number of branches decreased, (iv) the leaf area decreased, (v) the number of flowers decreased, (vi) the size of gall increased, (vii) the number of galls increased, (viii)the number of mature females per gram root increased, (ix) the size of mature female decreased, {11}
(x) the number of egg masses per plant increased, (xi) the amount of abnormal xylem increased, (xii) the amount of abnormal phloem increased, (xiii) the number of giant cell complexes per gall increased, (xiv) the size of giant cells decreased, and (xv) the giant cell cytoplasm evacuated more rapidly.
Different varities tested against root-knot nematode infection on the basis of plant growth, anatomical studies of roots, development and reproduction of the nematode, size of the mature female and the number of egg masses per plants, were categorized as follows :
1. Faizabadi Tolerant 2. Jhalarwali Moderately Tolerant 3. PDM Susceptible 4. Jaunpuri Susceptible 5. Baramasi Hyper Susceptible 6. Aligarh local Hyper Susceptible
Anatomical studies of infected roots carried out at regular intervals of time revealed the following facts : (i) the juveniles penerated the roots at or behind the root caps, (ii) the juveniles migrated inter-and intracellularly in the roots, (iii) After 24 hours, the juveniles reached meristematic zone, (iv) The juveniles caused hypertrophy, (v) Incipient giant cells with large nuclei observed. ( 12 }
(vi) After 48 hours, giant cells became prominent, (vii) The giant cells occurred in the differentiating phloem, (viii) After three days, hyperplastic tissue was observed, (ix) Hyperplasia and hypertrophy caused disruption in vascular strand, (x) The giant cells enclosed dense cytoplasm, (xi) After six days, abnormal xylem was observed, (xii) After 12 days, the giant cells enclosed very dense cyloplasm, (xiii) Amount of abnormal xylem increased, (xiv) After 18 days, small giant cells became empty, (xv) Empty giant cells transformed into abnormal vessel elements. (xvi) After 24 days, more abnormal xylem was seen. (xvii) After 30 days, more giant cells became empty. (xviii) Larger empty giant cells transformed into abnormal vessel elements, (xix) Juveniles hatched out of eggs, (xx) The juveniles caused secondary infection. nou9lea aement
1 express my sincerest gratitude and indebtness to Dr. Ziauddin Ahmad Siddiqui, Professor Department of Botany, Aligarh Muslim University, Aligarh who inspired me to initiate the research work in the fieldo f Plant Nematology especially Histopathology 1 am highly indebted to him for his keen interest, constant encouragement, magnificient devotion and fervent supervsion during the preparation of this manuscript
] am indebted to Prof. Saeed A. Siddiqui, Chairman, Department of Botany for providing me all Hbrary and laboratory facilities
I am also thankful to Prof. M. Wajid Khan, Ex-Chairman, Department of Botany for providing me the necessary facilities.
1, with insufficient and incompetent words, express my heartfelt sense of gratitude to Dr. Hisamuddin, Lecturer, Department of Botany, A.M.U. Aligarh for constructive criticism and valuable suggestions and sparing his precious time for going through this manuscript
Financial assistance from Aligarh Muslim University as J R F is also acknowledged
My humble thanks are also due to my parents, brother and sisters and Mr Nazir A Bhat, for their abundant support, patience, understanding and with whose encouragements and sacrificial devotions 1, ascribe all my success
Special thanks are extended to my friends and colleagues M Imran, Dr M Fazal, Kunhalvi, Showkat Ahmad, Dr Ashaq, Dr. Fahmida Hasan, Nikhat Yasmeen and Shazia Siddiqui for their whole hearted co-operation and encouragements
.Aisa Computers is thankfully acknowledged for the timely preparation of this manuscript
Finally, I bow in gratitude to ALMIGHTY ALLAH who provided and guided all the channels to work in cohesion and coordination and led me to this path of success
(Mohd. Yaqub Bhat) STUDIES ON HISTOPATHOLOGICAL TRANSFORMATIONS INDUCED BY MELOIDOGYNE INCOGNITA IN BITTERGOURD, MOMORDICA CHARANTIA
THESIS SUBMITTED FOR THE DEGREE OF Doctor of Philosophy IN BOTANY
MOHD. YAQUB BHAT
DEPARTMENT OF BOTANY ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA) 1999
Aligarh Muslim University, Aligarh eJ^lauddln f4. (Zjlddiqui M. Sc, Ph. D. (Alig.) Section of Plant Pathology and Nematology Department of Botany Professor of Botany Phone : 0571 401016 Ext 311,312 Int DatedJ.kr.LL'JJ.
TO WHOM IT MAY CONCERN
This is to certify that the work embodied in this thesis entitled "Studies on Histopathological Transformations Induced by Meloidogyne incognita in bittergourd, Momordica ctiarantia" is the bonafide work carried out by Mr. Mohd. Yaqub Bhat under my supervision and is suitable for submission for the Ph.D. Degree in Botany of Aligarh Muslim University, Aligarh.
PROF. ZIAUDDIN A. SIDDIQUI CONTENTS
Page No.
INTRODUCTION 1-20
REVIEW OF LITERATURE 21-59
SECTION-I 60-112
Experiment 1. 60-87 Experiment 2. 88-112
SECTION - II 113-148
Experiment 3. 113-131 Experiment 4. 132-148
SECTION - III 149-180 Experiment 5. 149-180
ibles 181-192
Photographs [1-87]
Summary 192-201
References 202-247 INTRODUCTION
Vegetables are rich source of certain essential vitamins, minerals, proteins and dietary fibres. The annual production of vegetables in India is estimated to be near about 72 million tonnes from about 14.4 million hectare area (Paroda. 1999). India being the largest producer of a variety of vegetables but the daily per capita consumption of vegetables is very low. The vegetables are one of the important items for export comprising of okra (Abelmoschus esculentus) bitter gourd {Momordica charantia) bottle gourd (Lagenaria siceraria) round gourd {Citrullus vulgaris XM festulosus), chillies {Capsicum spp^ green peas {Pisum sativum) cauliflower, (Brassica oleracea var botrytis) radish {Raphanus sativus) pointed gourd (Trichosanthes dioica) and sponge gourd {Luffa aegyptica). It means that the cucurbits are the main export vegetable items.
Cucurbits are of trailing habit and are cultivated during summer and rainy seasons. The family Cucurbitaceae includes about 90 genera and 700 species. Majority of the species are herbaceous and annual climbers. Fruits of cucurbits are extensively used as vegetables. Fully mature fruits are also used as fresh or for preparing jams and sweets, or fermented to give beverages. The roots of bitter gourd are astringent and are used medicinally. They are applied in bleeding piles, bowel affections and urinary complaints. The roots are pasted and applied over the body as sedative in fever. Fruits and leaves are used in external application for lumbago, ulceration and fracture of bones. (Sastri, 1962). The fruits and leaves of the plant contain two alkaloids, one of them being momordicine. Roots are also bitter. The plant is reported to contain a glucoside, a saponin like substance, a resin with an unpleasent taste, an aromatic volatile oil and a mucilage. The fruits, leaves and roots have long been used in India as a folk remedy for diabetis mellitus. The fruits are considered tonic, stomachic, carnminative and cooling. They are used in rheumatism, gout and diseases of liver and spleen. The fruits of uncultivated forms are used as febrifuge (Sastri-1962).
The soil harbours a large number of plant pathogenic organisms such as bacteria, fungi, actinomycetes, insects, and nematodes. In more recent years nematode diseases of cucurbit crops have increased their economic importance greatly. The root-knot disease caused by Meloidogyne spp. is one of the most economically important and cosmopolitan nematode disease of cucurbits in India. The pest usually attacks the underground parts of the plants where it induces the development of abnormal growth in roots. Root galling induced by Meloidogyne spp. is a well known host response of susceptible plants. Sometimes usually large galls are developed at the base of the stem. The size and character of these enlargements vary in different plants as in Thunbergia laurifolia and rhubarb enormously large structures, nearly two feet in diameter, may be seen (Steiner et. al, 1934).
Pathological effects of nematode feeding on crop plants range from simple mechanical injury caused by migration of the nematodes between or through plant cells, to complex host-parasite interactions. These plant and nematode interactions cause morphological and physiological changes of the affected tissues in plants. These changes involve damage or death of the cells by removal of their contents; or the host cells adapt to nematodes by enlarging and increasing their metabolic activities; or the cells undergo growth and multiplication. These effects of nematode parasitism of plants have been termed as destructive, adaptive and neoplastic, respectively (Dropkin, 1980).
Root-knot nematodes are sedentary endoparasites. The female nematode remains wholly embedded in the vascular cylinder. The sedentary life style is associated with distinct sexual dimorphism (Triantaphyllou, 1960; Davide and Triantaphyllou, 1967; Cohn and Spiegel, 1991). Second- stage juveniles usually invade or penetrate the roots in the elongation zone close to root meristem i.e; near the root tip. The nematode select either an epidermal cell or the site between two cells where the cell walls are still rather thin, (Wyss. et. ah, 1992). The movement of juveniles within roots is primarily intercellular (Nemec, 1910; Endo and Wergin, 1973). Intracellular migration has also been noted. (Christie; 1936).
The term giant cell refers to the multinucleate transfer cell, usually induced by root-knot nematodes in which the multinucleate condition of each giant cell results from repeated mitosis without cytokinesis. (Endo, 1987). Infection with root-knot nematode stimulates the formation of variable number of discrete giant cells (usually about 6 but reported to vary from 2-14) in host tissue. Hyperplastied and hypertrophied cells often surround the region of infection causing terminal and subterminal galling in infected roots. Kostoff and Kendall (1930) believed that giant cells were the result of cell wall dissolution of affected cells followed by coalescence of cell contents. This concept was supported by Christie, (1936); Krusberg and Nielsen, (1958); Dropkin and Nelson, (1960); Littrell, (1966). Huang and Maggenti (1969) in their detailed study on giant cell development in Vicia faba roots, found no evidence of cell wall dissolution of affected cells or coalescence of cell contents or cytoplasm in stimulated cells, they observed that multinucleate condition of giant cells arose from repeated endomitosis without cytokinesis of a single diploid cell. The metaphase chromosome number of giant cells in Meloidogyne javanica infected Vicia faba roots followed the geometric progression and not the arithmatic progression. They suggested a formula N=2d x 12 to predict the number of chromosomes in the giant cells in V. faba, where N represents total number of chromosomes, d total number of mitotic cycles, and 12 the diploid number of chromosomes. In a giant cell the nuclear changes range from a nucleus having one severely hypertrophied nucleolus to nuclei with various stages of membrane deterioration and lobulated periphery. They are generally irregularly lobed with a tremendously increased surface area which results in common linkages, between neighbouring nuclei.
The cytoplasm of a young giant cell becomes dense where golgi apparatus, mitochondria, ribosomes, polysomes and endoplasmic reticulum are abundant Central vacuoles gradually disappear and smaller vacuoles increasingly prevail (Jones and Northcote, 1972; Jones and Dropkin, 1976; Jones and Gunning, 1976; Jones and Payne, 1978; Wergin and Orion, 1981).
Synchronous nuclear divisions within the same giant cell have been observed for many other host plants (Krusberg and Nielsen, 1958; Bird, 1961; Owens and Specht 1964; Smith and Mai, 1965; Huang and Maggenti, 1969) but corresponding daughter cell wall formations have never been detected. However synchrony of nuclear division may not be constant (Huang and Maggenti, 1969; Bird, 1973). The polyploidy condition of giant cell commonly reaches to 32n and 64n (Huang and Maggenti, 1969).
Nuclei within giant cell are highly variable both in size and shape (Huang and Maggenti, 1969; Yousif, 1979). Other nuclear aberrations include nucleolar fragmentation so that small granules, stained like nucleoli remain scattered throughout the nucleus. Irregularly shaped, dumbell or sickle shaped nuclei have been reported by many workers (Krusberg and Nielsen, 1958; Owens and Specht, 1964; Rubinstein and Owens, 1964; Huang and Maggenti, 1969a). Nuclear enlargments result from swelling and in some cases from nuclear fusion where some of the nuclei attain a diameter of 35}i as compared to the normal cell nucleus of 6|im. The nuclear volume may increase to 10-12 times in tomato. (Rubinstein and Owens, 1964). Siddiqui and Taylor (1970) observed one or more vacuoles in the hypertrophied or fragmented nucleoli. Nuclear enlargement in giant cell of root-knot nematode infected tomato, cucumber and hawks-beard {Crepis capillaris) appeared to result from swelling and in some cases from nuclear fusion (Owens and Specht, 1964).
Christie (1936) found more dense cytoplasm near the head region of the nematode than remainder of the giant cell. As the nematode matures and nutrient demand from giant cell increases, the giant cell cytoplasm shows regions of intense metabolic activity. The giant cells bear appendages which grow intrusively among the neighbouring cells. Wall ingrowth or protuberance formation in giant cells may indicate a form of transfer cell. Sharma and Tiabi (1989) and Datta et. al., (1991) reported the giant cell formation in 72 hours after inoculation in the xylem and phloem parenchyma in Vigna radiata and Cyamoposis tetragonaloba.
Ediz and Dickerson (1976) found most of the giant cells in phloem region. The root-knot nematode readily penetrates the plant root within 24 hours after inoculation (Siddiqui and Taylor, 1970). However, other regions of the root are not immune to attack (Christie, 1936). Once inside the root, the migration occurs both inter and intracellularly (Nemec, 1910; Krusberg and Nielsen, 1958; Christie, 1936). Second-stage juveniles of nematodes penetrated freely into the epidermis and reached the endodermal layer intercellularly through the cortical cells in wheat. The cells surrounding the feeding site were darker and thicker than normal cells. Giant cell formation initiated mostly in phloem parenchyma and rarely in pericycle and xylem parenchyma in wheat seedlings, Patel and Patel (1991).
Galls are pathologically developed cells, tissues, or organs of plants that have arisen mostly by hypertrophy and hyperplasia under the influence of parasitic organisms. Formation of giant cell and multiplication of pericycle and other parenchyma cells around the nematode head cause the vascular tissues to divert from their normal path. Xylem and phloem also becomfe abnormal with reference to structure and orientation. Small as well as hypertrophied parenchyma cells are transformed into vessel like elements and constitute the abnormal xylem. Since giant cells are highly metabolically active cells, therefore, they must directly or indirectly be connected with vascular tissues.
Galling is one of the earliest host response in root-knot nematode infection in the roots of host plant. Molliard (1900) observed galls on the root of melon, Coleus and Begonia and reported that after invasion, the root tip growth may be arrested and lateral roots frequently developed near the site of invasion.
Schuster and Sullivan (1960) found galls in tomato by Meloidogyne incognita larvae even when they did not enter the roots. The stylet penetrated the root surface cells and secreted materials that stimulated host tissue to form galls. Davis and Jenkins (1960) reported gall formation in Gardenia spp. infected with M incognita, M. incognita acrita and M. hapla. Cortical and stelar proliferation accompanied all infections. M javanica infection on soybean roots caused hypertrophy, hyperplasia and giant cell formation in the tissue surrounding the head that consequently led to gall formation (Ibrahim and Massoud, 1974). According to Siddiqui and Taylor (1970) gall formation is attributable to hypertrophy of the cortical and egg mass production.
Huang (1966) reported cork and lignified wall thickening of endodermis and pericycle at infection sites on Zingiber officinale. Division of stele in root galls of Lycopersicon pimpinellifolium infected with M incognita was observed by Farooq, (1973). Root-knot nematode stimulated the cells of pericycle to divide, forming parenchymatous outgrowths which grew into lateral roots. Some times the inner most parenchymatous cells become differentiated into xylem elements of irregular shapes. Siddiqui et. al., (1974) reported reaction xylem formation in Lagenaria infected with root-knot nematode. Pasha et. a/., (1987) observed irregularly scattered vessel elements that caused discontinuity of vascular tissues and significant reduction in vessel dimension. Hypertrophy, hyperplasia, thickening of cell walls, granular cytoplasm and enlarged nucleus and nucleoli were reported around the nematode head.
Attractiveness of Meloidogyne spp. juveniles was observed towards the root and excised shoot tissue of several host plants (Linford, 1939). Griffin and Waite, (1971) reported attraction of M incognita juveniles towards both resistant and susceptible alfalfa seedlings, when an egg mass w as placed mid way between germinating seedlings. Susceptible alfalfa plants have a stronger attraction to M. hapla larvae than do resistant plants leading to higher infection occurring in susceptible than in resistant plants (Griffin, 1969). M. hapla larvae were found attracted towards the seedlings and excised root tissue of susceptible tomato, beans, eggplant, and soybean (Lownsbery and Viglierchio, 1961). Fast growing tomato plants attracted M. hapla more strongly than slow growing plants. In resistant varieties the juveniles of root-knot nematode either do not enter into the roots or if enter the> do not develop properly (Barrons, 1939; Christie, 1949; Sasser and Taylor, 1952). Resistance in certain plants toward root-knot nematode may develop with age (Griffm and Hunt, 1972).
Godfey and Oliveira (1932) reported that penetration in cowpea and pineapple roots by root-knot nematode occurred in less than 6 hours. Single and multiple larval penetrations were readily detected. Migration in root tissues continued upto three days until the nematode became sedentary. Root-knot nematodes penetrated through ruptured tissues of sweet potato roots caused by secondary root emergence and at abnormal openings such as cracks in the surface of enlarged roots (Krusberg and Nielsen, 1958). Roman (1961) reported galleries and burrows of broken and separated cells that indicated intracellular penetration by juveniles.
Hypersensitivity is a common response of resistant plant (Rohde, 1965) in which the larvae may enter the roots of resistant plants in large numbers but hypersensitive cells quickly die and wall off the pathogen so that injury to host remains confined to a few cells. (Riggs and Winstead, 1959). This results in the subsequent death of nematode. The hypersensitive reaction in some cases may be delayed. Linford (1939) reported penetration, development, and migration of cotton root-knot nematode, Meloidogyne incognita acrita in resistant and susceptible alfalfa varieties. He found no development of nematode in resistant cultivar but egg laying was observed after 18 days of penetration in susceptible cultivar. The larvae readily enter resistant as well as susceptible plants in approximately the same number (Riggs and Winstead, 1959; Williams, 1956). However, contrary to that the number of M hapla larvae entering resistant tomato plants was less in resistant than in susceptible plants (Dropkin and Web; 1967).
Christie (1949) found that cortical and vascular tissues of resistant plants surrounding the parasites become vacuolated and fail to be served as food. The resistant plants do not always develop cell necrosis in response to nematodes (Dropkin, 1969). Veech and Endo (1970) during their study 10 suggested that, soon after the penetration of root-knot nematode in soybean roots, enzymatic activity is slightly increased at the feeding site in both susceptible and resistant plants. Giant cell induction occurred in the susceptible host and cell necrosis was found in resistant host. In a resistant variety of cotton, McClure et. al., (1974) observed immobilization of M incognita larvae, their inability to induce giant cells, and finally their death and disintegration within the roots.
Meloidogyne incognita acrita larvae penetrated equally in three species of cucumber. They developed more rapidly on Cucumis melo than other species. Resistance in C.ficifolius and C. metuliferus was associated with hindrance of larval development beyond the second stage, delayed development of larvae to adults, and stimulation towards maleness. Tissue necrosis or hypersensitivity was not associated with larval penetration. In C. metuliferus both normal giant cell and small giant cells were found. The nematodes associated with small giant cells were immature. Cellular stimulation was limited to a few cells near the head that caused a limited amount of hypertrophy (Fassuliotis, 1970). West Indian gherkin (C. anguria) and few other species of Cucumis were found resistant to M incognita andM arenaria (Fassuliotis and Rau, 1963; Fassuliotis 1967).
Thirty two auto-and allotetraploids were used as possible sources of resistance in some perennial Triticeae to M chitwoodi, all were found resistant to it by Jensen and Griffin (1997). Gall rating was found low with significant P<0.01 difference among accessions of the same species, among species, and among genera with different genomes. Significant difference 11 was found in reproductive factor among species with same genomes, but no difference in reproductive factor among species with same genomes, but no difference in reproductive factor among genera with different genomes and accessions within the same species and genome.
In resistant plants, Dropkin and Webb (1967) found unusual responses towards Meloidogyne incognita acrita and M hapla. Some plants had no galling response others had small, inconspicuous galls that were few in number (Fassuliotis et. aL, 1970; McClure et. al., 1974). Nematodes, however did not reach to maturity (Fassuliotis and Dukes, 1972; Golden and Shafer; 1958). Canto-Saenz (1984) found no detectable traces of juveniles but observed only necrotic cells. Lastly the nematode disintegrated and disappeared. (McClure et. al., 1974).
Dropkin (1969) found that incompatible plants are not always hypersensitive (Giamalva, et. al., 1963; McClure et. al, 1974). As juveniles lie closely appressed and parallel to the steles, giant cells, if formed may be abnormal and not fully developed and the nematode either produces no eggs or few eggs (Christie, 1949; Dropkin and Nelson, 1960; McClure, 1974). The extent of giant cell development is related to the degree of nematode development (Endo, 1965;Rohde, 1972).
Nematodes generally locate and penetrate roots of most of the compatible and incompatible plants equally (Huang, 1986; Ibraham et. al., 1980; Raja and Dasgupta, 1986). Pedrosa et. al, (1996) reported penetration, post infection development and reproduction oiMeloidogyne arenaria races 1 and 2 on susceptible and resistant soybean genotypes. More juveniles 12 penetrated the roots of susceptible cultivars than the resistant genotypes but the ability to locate and invade roots was similar between races. There was a significant difference in development of race 1 and 2, after 10 days in susceptible and resistant genotypes. Female development of race 2 initiated after 20 days in all genotypes. Fewer eggs per root system by race 1 were seen than race 2 after 45 days of inoculation.
While evaluating resistance and susceptibility in wild Solanum spp., Janssen et. al., (1996) observed large differences in number of juveniles of M. hapla and M fallax in soil between resistant and susceptible genotypes. Moreover, at the end of growing season, the level of infection in soil for all resistant genotypes was equal or at lower level than at the beginning of growing season.
Soil fumigants and non-fumigants are used as nematicidies for the control of nematodes, non-fumigant nematicides have several advantages over fumigants which sometimes outweight their greater basic cost. They are generally much less phytotoxic. They require no special equipment and are easy to apply in soil with lower dosage rates to control nematodes effectively. Non-fumigant nematicides have less persistent residues (Van Berkum and Hoestra, 1979). Nematicides reduce the movement, invasion, feeding and consequently the rate of development and reproduction. (Evans, 1973; Nelmes et. al., 1973). Nematicidal potency necessarily does not depend on nematicide accumulation in nematode, but sensitivity of the target site to different pesticides is also important.
Aldicarb, a relatively hydrophilic nematicide accumulates in 13 nematodes to a lesser extent and thus a better nematicide than the more lipophilic pesticide phorate (Batterby et. al., 1977). Considerable differences of the organophosphate and carbamate pesticide effects on different stages of nematode life cycle have been reported.
Invasion of plants by plant parasitic nematodes is reduced, and the nematode development is inhibited in plant on application of organophosphates and carbamate nematicides (Whitehead, 1973; Bunt, 1975; McLeod and Khair, 1975; Wright et. al., 1980).
Wright and Womack (1981) reported significant inhibition of development of M incognita juveniles at 6 ppm oxamyl concentration in cucumber roots. It also inhibited the development, of other species of Meloidogyne juveniles, to swollen forms for at least 30 days following application of the pesticides. Direct orientation ofM incognita juveniles towards cucumber roots was shown to be inhibited at the concentration, (0.5) of oxamyl. However nematode movement was not affected at this concentration, large number of individuals eventually reached the roots but there was significant reduction in number of juveniles invading the roots (Wright et. al., 1980). Oxamyl treatment of tomato seedling cotyledons or stems reduced the number of invading M javanica juveniles around the roots.
Time taken by individual nematodes to attack roots was found to increase. Organophosphate compounds and carbamate nematicides have been found to reduce fecundity in a number of species (Myers, 1972; Wasilewska, 14 et. al., 1975; Atilano and Van Gundy, 1979; Van Berkum and Hoestra, 1979). DBF has been found to reduce the number of egg masses and eggs produced by M. incognita (Veech, 1978a) and population of several species (Veech, 1978b), with no apparent juvenile toxicity.
Nematicide movement and other exogenous compounds in plant is either apoplastic or ambimobile. Mostly there is symplastic movement of systemic insecticides and nematicides. A few such compounds are ambimobile with some symplastic movements (Crafts and Crisp, 1971; Crisp, 1971; Bunt and Noordink, 1977).
As the nematodes are not directly killed by the chemicals, it is difficult to distinguish between a direct contact action in the soil water and a real systemic action via the plant. Inside a plant, nematodes can be influenced either by direct contact action or by an indirect effect resulting from ingesting the poison when feeding on the cell contents. Penetration, feeding, and reproduction of nematodes in or on the plant tissues can be influenced by direct contact action via the soil water as well as by a systemic action via the plant or by both. (Bunt 1975; Evans and Wright, 1982; Marban- Mendoza and Viglierchio, 1980). Pankaj and Siyanand (1992) reported the efficacy of chemicals as seed dresser against A/, incognita in bitter gourd and round gourd. It caused the inhibition of larval penetration, female development, and gall formation on bitter gourd and round gourd.
Plants develop close associations with many soil organisms specially with fungi and nematodes. These organisms independently, develop 15 associations with plants that are either beneficial or harmful to the plant and either facultative or obligate for organisms. Fungi appear to act as biological control agent of Meloidogyne spp. but often this is difficult to demonstrate convincingly (Al-Hazmi et. al; 1982). Although several fungi, bacteria and sporozoans are known to reduce nematodes populations under laboratory and green house conditions, results of field trials have been inconclusive and for most cases, disappointing. The fungus Paecilomyces lilacinus. (Thom) Samson has been reported as a potential biocontrol agent for root-knot nematode. (Jatala et. al, 1979; Jatala, 1982, 1986; Adiko, 1984;). It consistently and efficiently controls the population of root-knot nematodes, {Meloidogyne spp.) (Jatala et. al, 1979; Jatala et. al, 1980; Jatala et. al, 1981). P. lilacinus a common hyphomycete closely related to Penicillium, (Samson, 1975) parasitieses eggs of Meloidogyne spp. (Dunn et. al, 1982; Jatala, 1986). P. lilacinus penetrates both eggs and females of Meloidogyne spp. (Jatala, 1982, 1986, Adiko, 1984; Freire and Bridge, 1985). However, the exact mode of parasitism is unknown.
Fungal colonization of giant cells and xylem is common in fungus root-knot interaction. Fattah and Webster (1983) observed normal giant cells and fungal infections in one week old inoculated seedlings of tomato cultivars. After two weeks, hyphae were visible in xylem tissues of Fusarium susceptible but not in resistant plants. Dissolution of giant cell wall occured where giant cells were in direct contact with hyphae. No such changes were seen in the giant cell of control plant inoculated with nematodes alone. Ryder and Crittenden (1965) found that drastic change in the development of M incognita acrita in cabbage roots infected by Plasmodiophora brassicae. 16
Giant cells were invaded by fungus and the nematode failed to devlelop to maturity due to disruption of feeding site.
Abundant fungal growth was observed in nematode induced giant cells as well as in xylem, in roots of cotton jointly infected by Fusarium oxysporum f sp. vasinfectum and M. incognita acrita. Better growth of fungus was observed in decaying cortical and epidermal cells but poor in healthy tissues. It entered the xylem through decaying tissue (Minton and Minton, 1963). Giant cells, extensively colonized by the fungus lost their contents soon after invasion by the fungus. Female nematode and egg mass invasion by the fungus was also witnessed. Fungal colonization was not restricted to only galled tissue but vigorous hyphae extended up into the xylem above the soil line Davis, 1963; Melendez and Powel, 1967). Powel (1971) reported some nonpathogenic soil inhabiting fungi including Penicillium martensii (close relative of Paecilomyces) causing invasion of roots previously exposed to M incognita with extensive decay. Cabanillas et. al., (1988) found no galling and giant cell formation in tomato roots inoculated with nematode eggs infected with Paecilomyces lilacinus. Few to no galls and no giant cell formation were observed in roots dipped in spore suspension of P. lilacinus and inoculated withM incognita.
Bitter gourd {Momordica charantia) is an annual climber, cultivated throughout India. Bitter gourd is grown as vegetable crop. Fruits and leaves have antihelmenthic property and are used to cure piles, leprosy, jaundice and as gastric vermicide. Roots are astringent, used in haemorrhoids, and applied over the body as sedative in fevers. 17
Momordica charantia is attacked by a large number of diseases of which nematode diseases are of economic importance. Root-knot nematode Meloidogyne incognita, an endoparasite, results in the formation of large sized galls on roots of bitter gourd. Giant cells are formed as a result of hypertrophic reaction caused by M. incognita that also lead to abnormal xylem and abnormal phloem formation. The continuity of vascular cylinder seems to be distrupted as a result of infection. The consequences of abnormalities in vascular elements lead to poor and stunted growth of plant and reduction in yield.
Since, the nematode is a devastating pathogen, therefore it needs to be managed. P. lilacinus a potential biological control agent of root-knot nematode colonizes the egg masses and reduces nematode population which helps in enhancing the plant growth. Although the consistent association of P. lilacinus with eggs of Meloidogyne spp. and its ability to penetrate both eggs and females is well known. The exact mode of its parasitism is unknown. There are few reports of histological examination of the fungus on Meloidogyne parasitising plants. Thus, it was felt desirable to carry out the histological examination of M incognita infected and P. lilacinus treated M. charantia plants. The objectives of this study were to examine (i) the effect of Paecilomyces lilacinus on M. incognita infected plant tissues, (ii) the effects of fungus on the nematode, (iii) the effects of fungus on the eggs and the egg masses, and (iv) the effect of fungus on the giant cells.
Similarly, aldicarb a systemic nematicide, persistent in soil, inhibits the development of Meloidogyne incognita and decreases the rate of 18 penetration and movement in soil. Till this date no mentionable work on histological examination of Meloidogyne infected and aldicarb treated plants has been carried out. Therefore it was felt necessary to carry out some work on histological tranformations in presence of nematode and the nematicide. The objectives of the study were (i) to investigate anatomy of the Meloidogyne incognita infected and aldicarb treated Momordica charantia plants (ii) to study the effects of aldicarb on the nematode at various stage of development (iii) to study the effects of aldicarb on eggs and egg masses, their hatching and on hatched juveniles and (iv) to study the effect of aldicarb on the giant cells.
Pathogenicity tests are important to determine the threshold levels of the pathogens and have significant bearing on the disease development in a given state of condition. As the effect of different inoculum levels is influenced by the number of ecological factors, therefore, it is necessary to determine the effect on disease development at various inoculum levels and its effect on plant tissues and giant cell formation. Available M. charantia varieties were histologically examined to see any difference in susceptible and resistant plants in presence of root-knot nematode, Meloidogyne incognita
Gall formation is the observable response of plant towards root-knot nematode infection. The galls are the result of hyperplasia and hypertrophy in the affected part of the root. The gall induction is initiated soon after penetration of the nematode. The nematode itself and the tissues affected by it lead to abnormalities in the galled region. 19
The objectives of this work were (i) to study the role of giant cells, and other hypertrophic and hyperplastic tissue in gall formation, (ii) to compare from earlier findings the anatomical changes in the formation of the giant cells and the galls, (iii) to investigate the origin oj^giant cells and their fate after death of the nematode, (iv) to find out any link of giant cells with the phloem, and (v) to observe abnormalities in xylem and phloem.
The experiments were performed to compare and confirm earlier reports and to provide some new information. For this purpose following experiments were conducted.
Section-I
Experiment 1. Effect of Paecilomyces lilacinus on histopathology of Meloidogyne incognita infected Momordica charantia plants.
Experiment 2. Effect of a systemic nematicide (aldicarb) on the growth, and histopathology of roots, of Momordica charantia infected with Meloidogyne incognita.
Section-II
Experiment l.The effect of different inoculum levels oi Meloidogyne incognita on Momordica charantia.
Experiment 2. Histopathological responses of selected varieties of Momordica charantia to Meloidogyne incognita. 20
Section-Ill
Experiment I. Origin of giant cells, development of galls and histology with special reference to xylem and phloem, of galled roots of Momordica charantia infected with Meloidogyne incognita. REVIEW OF LITERATURE
Root-knot nematodes, Meloidogyne spp. are the most important and the best known nematodes. They have evolved very specialised and complex host-parasite relationship. Meloidogyne spp. parasitise a wide variety of host plants belonging to monocotyledons and dicotyledons including both herbacious and woody plants. Meloidogyne spp. cause the formation of familiar knots or galls on the roots of susceptible host plants resulting in severe growth retardation of plants.
Root-knot nematodes are known to parasitise more than 2000 species of plants, but host-parasite rotationships have been investigated only in few plants (Webster, 1969, 1975; Taylor and Sasser, 1978 and Hussey 1985). The first record of any nematode injury of vegetables was that of root-knot nematode damage to cucumber in an English green house (Berkeley, 1855). Root galling induced by Meloidogyne spp. is the well known host response which involves the production of abnormally large multinucleate cells known as giant cells, in the vascular tissues of susceptible plants. Generally, root damage caused by parasitic nematodes is reflected on the above ground portion of the plant as poor shoot growth, leaf chlorosis and even death of plants resulting in low yield and poor quality produce.
The stresses inflicted upon by the nematodes on the plant are manifested in the form of lesser tillering, yellowing and stunting of plants which result in low productivity. The above ground symptoms are similar to those associated with any root injury that result in reduced amounts of water 22 uptake by plants. Flowering is scanty and fruits are either lacking or are of poor quality (Jenkins and Taylor, 1967). Meloidogyne spp. also interfere with the process of nitrogen fixation in the nodules of leguminous plants.
In Meloidogyne spp. the second-stage juvenile is the first stage that infects the plants and starts its life cycle on susceptible host. The juveniles penetrate the root tips (Christie, 1936) and feed on root hairs and epidermal cells. They accumulate either at the region of cell elongation just behind the root cap, or at the points where lateral roots emerge, or at Ihe site of penetration of other juveniles and cut surfaces of roots (Godfrey and Oliveira, 1932; Linford, 1939, 1942; Peacock, 1959, Bird, 1969; Green, 1971; Siddiqui, 1971a, b; and Prot, 1980). The mechanism of penetration may involve mechanical action by thrusting of stylet or cellulolytic and pectolytic activity of enzymes (Linford, 1942; Bird and Loveys, 1980). Primary root penetration by second-stage juveniles occur at the tips of the young roots in the region of differentiation in sweet potato or anywhere from root hair formation and also through the loose ruptured cells of enlarging tuberous roots (Krusberg and Nielsen, 1958). Siddiqui and Taylor (1970) found most of the juveniles penetrating in the region of cell differentiation and elongation. After penetration intercellular migration of juveniles in the cortex was detected (Nemec, 1910, Godfrey and Oliveira, 1932; Linford, 1937, 1942; Endo and Wergin 1973; Jones and Payne, 1978). During intercellular migration the cells became separated along the middle lamella. Along the path of migrating juveniles, the cells distented and compressed and did not show any sign of rupture or nematode feeding. Inter- 23 and intracellular migrations were reported by Christie, (1936); Knisberg and Nielsen,(1958); Roman, (1961); Bird, (1959, 1960, 1962); Siddiqui and Taylor, (1970); and Siddiqui, (1971a,b). Roman (1961) observed galleries and furrows of broken and separated cells that indicated intracellular penetration by juveniles.
Giant cell is a multinucleate transfer cell in which the multinucleate condition results from multiple mitosis in absence of cytokinesis. Formation of giant cell is not accompanied by cell wall dissolution (Endo, 1987). Giant cells are highly specialised cellular adaptations induced and maintained in susceptible hosts by the feeding oiMeloidogyne spp. Primary phloem or adjacent parenchyma are the highly preferred tissues for the development of giant cells (Christie, 1936; Krusberg and Nielsen, 1958; Byrne, 1977). Beille (1898) was the first who proposed that the giant cells are formed by the disintegration of cell walls and coalescence of cells in Papaya roots. Observation of cell wall fragments in giant cell cytoplasm strengthened this view (Kostoff and Kendall, 1930; Christie 1936; Krusberg and Nielsen, 1958; Dropkin and Nelson, 1960; Roman, 1961; Krusberg, 1963; Bird, 1961, 1972, 1974; Owens and Specht, 1964; Birchfield, 1965; Smith and Mai 1965; Littrell, 1966; Siddiqui and Taylor, 1970; Rohde and McClure, 1975). The other view emphasised cell enlargement and endomitosis which was supported by a number of researchers (Myuge, 1956; Huang and Maggenti, 1969; Paulson and Webster, 1970; Jones and Northcote, 1972: Jones and Dropkin, 1976; Jones and Payne 1978). The rate of increase in numbers of nuclei for all plant species was greatest during the first seven days of inoculation. No mitotic activity was observed in the gaint cells 24 associated with adult nematodes.
Dropkin (1965) reported polyploidy in giant cell of hairy vetch induced by a Meloidogyne species. More than 2N chromosomes occurred within giant cells and in surrounding cells of hairy vetch (Vicia vellosa), 6 days after invasion. Synchronous nuclear divisions within one giant cell but not in other of the same set were observed. Some nuclei displayed over 100 chromosomes than normal (2N=14). Synchronous nuclear divisions were also reported by Bird (1961).
Huang and Maggenti (1969a, b) observed giant cells in the roots of Viciafaba infected with Meloidogyne javanica, formed by repeated mitoses of the original diploid cell without subsequent cytokinesis. They reported chromosome number as 4n, 16n, 32n and 64n. Interphase nuclei with many chromosomes were formed due to fusion of mitotic apparatus in giant cells either at metaphase or anaphase stages. Many interphase nuclei, however, were simply in close juxtaposition instead of actually being fused as revealed by electron microscopy. The interphase nuclei were irregularly lobed and frequently assumed amoeboid appearance. Mitoses in giant cells were generally synchronised. Some nuclei in hyperplastic tissue around giant cells were also lobed and definitely smaller than those of giant cells. The cell plate formation was not observed from binuclear stage upto the multinuclear stage in a giant cell. They did not find any evidence of cell wall dissolution. Synchronous nuclear divisions within the same giant cell without daughter cell wall formation have been observed in many plants; (Krusberg and Nielsen 1958; Bird 1961; Owens and Specht, 1964, Smith and Mai, 1965). 25
Root-knot nematode {Meloidogyne spp.) after establishing a permanent feeding site in the host roots induced the formation of enlarged symplastic structures called giant cells (Jones and Northcote, 1972a). The giant cells have the ability to transfer metabolites from the host to developing larvae. There are ample evidences indicating that transfer cells are metabolically hyperactive cells and essential for the development of the nematode. (Bird, 1961, Littrel 1966; Endo and Veech, 1969a, Veech and endo, 1969; Webster, 1969; Endo, 1971; Dropkin, 1972; Gommers and Dropkin, 1977). Hussey and Sasser (1973) observed peroxidase in the style exudate of M incognita females and suggested that this enzyme is involved in giant cell induction and maintenance.
Giant cell development and their maintenance in plants infected with Meloidogyne javanica depended on continuous stimulus from the nematode whose removal resulted in break down of the giant cell (Dropkin, 1954). Normal giant cell development took place under sterile conditions. A growth promoting substance was traced in galls but not in adjacent roots. Roots of heavily infected plants were so severely damaged that individual galls coalesced into amorphous masses containing large number of nematodes. It was noted that galls on tomato roots were all of about the same size, and number of larvae within a gall could be predicted by measuring the size of the gall.
Malformation of galled roots on bent grass was reported by Hodges et. al., (1963); Hodges and Taylor, (1966) and on edible ginger by Huang, (1966). In edible ginger, wound cork was observed at feeding site. The 26 mature females laid egg masses in internal lesions, where the larvae hatched from fresh egg masses were trapped.
According to Krusberg and Nielsen (1958) the young root tips, lateral root ruptures, and the surface of cracks were the three major infection courts in sweet potato roots. After penetration, the second-stage larvae migrated inter-and intracellularly to the feeding site that varied with the infection court. Finally the larvae came to rest, primarily in the stele, in the region of cell elongation or the cambial zone. Nematode feeding stimulated the formation of several atypical tissues viz., giant cells, abnormal xylem, hyperplastic parenchyma and cork.
Penetration leading to intracellular migration in wheat roots causing cortical hypertrophy occurred within 24 hours after inoculation of M naasi. The giant cells developed within 4-5 days around the head of each nematode in the stelar region. Initial pathological alterations due to giant cell formation were found in protophloem and protoxylem. Giant cells contained 2-8 agglumerated multinucleolate nuclei. Synchronous mitotic division was first observed 9 days after inoculation. After 21 days giant cells became highly vacuolated. Observations, 40 days after inoculation, revealed complete degeneration of cell contents in many giant cells, but their thick walls remained intact. Abnormal xylem completely surrounded the degenerated or partially degenerated giant cells (Siddiqui and Taylor, 1969). A large number of M. naasi larvae penetrated root tips of wintock oats within the first 24 hrs, and the first sign of incipient giant cell formation was visible four days after inoculation (Siddiqui, 1971). Three types of 27 responses were observed by him: (a) necrosis of inner cortical and endodermal cells in contact with the nematode lip region and failure of larvae to enter the stele, (b) necrosis of cells around the incipient giant cells resulting in degeneration of giant cells and larvae, and (c) uninterrupted giant cell development which is not distinguishable from that in susceptible host.
Jones and Payne (1978) observed giant cell induction in roots of Impatiens balsamina by Melofdogyne spp. under light and electron microscope. They reported that first sign of giant cell formation was the division of cells around the larval head. Cell plate alignment appeared to proceed normally but cytokinesis failed resulting in the formation of binucleate cells subsequently; no wall breakdown was evidenced. The number of nuclei increased due to repeated mitoses without cytokinesis. Nuclear division was detected 24 hours after inoculation in the cells upto two cell layer from the head of the nematode. After 48 hours, 2,4 and 8 nuclei were observed in the cell near the nematode head. In early stages of giant cell formation, cell wall breakdown and cell fusion were not evidenced under electron microscope, wall gaps or holes in a continuous cell wall were not detected. Cell wall of a giant cell consisted of thick and thin areas. The area of the cell wall where plasmodesmata occur is close to the limit of resolution of light microscope. This gives an impression of gaps in the wall. The irregular outline of individual giant cell at many sites due to cell wall ingrowth, appeared as tylose like structure which, according to Jones nd Payne (1978), was considered by certain workers as cell wall fragments. In binucleate cells, 48 hours after inoculation the cell plate vesicles aligned 28 normally but dispersed without fusion and thus failed to form cell plate and consequently daughter cell wall. The cell plate vesicles were also thought to be the fragments of broken walls. In Glycine max, M. javanica caused giant cell formation after 21 to 32 days of inoculation (Subbotin, 1990).
In sorghum roots, on infection with cotton root-knot nematode, the development of the giant cells in the cortex or stele of lateral roots was studied by Orr and Morey (1987), the giant cells developed either singly with few nuclei or in groups with many nuclei. Interruption of pericycle and endodermal tissue was not observed upto the one third of the circumference of stele. In the area where pericycle and endodermis were absent the parenchyma of the cortex extended to the vascular elements and abnormal xylem around the giant cells extending into the cortex. The galls appeared on sorghum roots as elongated swellings, discrete knots, or swellings with root proliferation.
Pasha, et al. (1987) while working on Solarium melongena infected with Meloidogyne incognita observed feeding site of nematode mainly in stelar region. The cells in the feeding site exhibited hypertrophy, hyperplasia, thickening of cell wall, granular cytoplasm and enlarged nuclei and nucleoli. Abnormal xylem, formed in response to infection, occurred in irregular patches causing discontinuity in vascular tissue, was also seen. Bilqees and Jabeen (1994) observed cavities in banana roots. The cavities originated by cell proliferation, cell wall destruction and lysis of cells in response to the pressure exerted and toxic materials excreted hy Meloidogyne spp.
Intercellular migration of Meloidogyne larvae, their feeding sites 29 being in ground tissues, and disruption of vascular elements have been reported by Fawole (1988) in white yam; Kim and Ohh (1990) in tomato; Sharma and Tiabi (1991) in pea; Patel and Patel (1991) in wheat, Salawu (1991) in Celosia argentia; Datta et. al., (1991) in Vigna cymopsis; Husain et. al., (1992) in tomato and brinjal. Hisamuddin and Siddiqui (1992) also observed intercellular migration of M incognita larvae, but they emphasized the feeding site to be in protophloem originating from procambium. They did not find disruption in vascular strands. Moreover, gaint cells were found to be actively connected with phloem strands. Vovlas and Sasnelli (1993) also reported cambium as the feeding site of Meloidogyne juveniles in the roots of Helianthus.
Meloidogyne juveniles penetrate the roots of many plants but only suceptible plants respond to feeding and undergo pronounced morphological and physiological changes. The histopathological changes that contribute in the formation of galls are (i) hypertrophy of cortex, xylem parenchyma, and metaxylem (ii) hyperplasia of pericycle and phloem parenchyma (iii) enlargement of nematode body and (iv) egg mass production (Christie, 1936; Krusberg and Nielsen, 1958, Dropkin and Nelson, 1960; Bird, 1961; 1962; Owens and Specht, 1964; Hodges and Taylor, 1966; Siddiqui and Taylor, 1970; Siddiqui 1971a, b; Swamy and Krishnamurthy, 1971; Prakaso Rao and Arunee 1973; Ibrahim and Massoud; 1974). Shephered and Huck (1989) reported small cracks in root epidermis and cortex by M incognita.
Meloidogyne generally interferes with the vascular system and causes extensive damage to xylem and phloem. Christie (1936) noted that trachieds 30 or vessels were interrupted in the gall region and passed around giant cells. The continuity of the vascular cylinder was, however, more or less maintained. Additional xylem elements of irregular shape were formed from cells of the surrounding parenchyma. Krusberg and Nielsen (1958) while working on sweet potato infected with root-knot nematodes discussed a lot about abnormal xylem but did not mention about the fate of normal vascular column. Cells of abnormal xylem were mostly devoid of contents, although a few contained nuclei. They were derived directly from xylem parenchyma and were characterized by secondary wall thickenings of annular, reticulate or pitted type. Their shapes corresponded to the shape of parenchyma cells from which they were derived, intercellular spaces, however, were lacking. The xylem and phloem in Gardenia was interrupted by giant cells, proliferated parenchyma cells and the nematode, which caused the vascular tissue to occur in irregular patches rather than in one continuous column (Davis and Jenkins, 1960). In balsam, Odihirin and Jenkins (1965) reported interruption in continuity of most vessels and other vascular tissues due to giant cell formation. Xylem and phloem tissues were scattered and appeared as patches at abnormal positions. Huang (1966) observed giant cells only on fibrous roots of Zingiber where all infections accompained abnormal xylem and hyperplastic parenchyma. In peony roots, the vascular tissues comprising of xylem and phloem were mostly parenchymatous and were having much starch (Eversmeyer and Dickerson, 1966). Swamy and Krishnamurthy (1971) found that the initial target of infection in Basel la was the primary phloem tissue in young roots, and secondary phloem or ray 31 tissue in older roots. There was either little or no injury to the primary or secondary xylem. Inspite of the ubiquitous differentiation of xylem as a consequence of infection, there was a total absence of phloem differentiation and even functional phloem was destroyed soon after infection.
A bifurcated stele was noticed in Lycopersicon pimpinellifolium infected -with Meloidogyne incognita (Farooq, 1973). Siddiqui and Ghouse (1975) observed that in early stages of Meloidogyne incognita infection on Lagenaria leucantha roots, the phloem was destroyed due to pressure of undifferentiated tissue produced by the cambium. After destruction of normal phloem, new phloem was developed which differed in orientation, composition, structure and size from the normal phloem. It mostly comprised of parenchyma and some sieve tube elements but no companion cells.
The abnormality in orientation of vascular strands started 48 h after inoculation. Vascular strands distorted severely due to multiple hypertrophic and hyperplastic reactions taking place continuously. The strands became wav>' and appeared as scattered patches, when seen in longitudinal sections. The vessle elements became structurally abnormal within 48 h of inoculation. The vessel elements broadened near the giant cells due to hypertrophic reactions. Irregular shapes and sizes of vessel elements of metaxylem strands were consequenced upon pressure exerted on them by the tissue resulting from hypertrophic and hyperplastic reactions. Abnormal origin of vessel elements was observed 72h after inoculation from small parenchyma 32 cell; 9 days after inoculation from hypertrophied parenchyma cells; 15 days after inoculation from small giant cells; and 30 days inoculation from larger giant cells. Formation of vessel elements in excess amount might increase upward translocation, or supply water to giant cells, or provide protection to giant cells, or give support to entire gall (Hisamuddin, 1992).
Nearly all the Meloidogyne /«cogw/7<7 juveniles selected the primary phloem or adjacent stelar parenchyma as feeding sites, after penetrating the primary roots of Glycine max (Bynie et. al., 1977). Because of the giant cell development, the normal phloem and xylem connection between the primary and lateral root was abolished. Not only were potential primary phloem and xylem connections incorporated into giant cells, but meristematic activity associated with gall formation precluded differentiation of a normal vascular connection. With continued nematode feeding, some of the cells destined to become phloem and xylem were converted to giant cells. Finley (1981) found Meloidogyne chitwoodi larvae embedded invariably in the phloem in potato roots. M incognita infection caused disruption in vascular strands of eggplant roots which resulted in breaking up of the vascular continuity. Abnormal xylem comprising of vessel like elements derived from stelar parenchyma was of universal occurrence associated with Meloidogyne infections (Christie, 1936; Krusberg and Nielsen, 1958; Davis and Jenkins, 1960; Odihirin and Jenkins, 1965; Eversmeyer and Dickerson, 1966; Huang, 1966; Littrell, 1966; Siddiqui and Taylor, 1970; Siddiqui, 1971a, b; Swamy and Krishnamurthy, 1971; Farooq, 1973; Siddiqui et. al., 1974; Ngundo and Taylor, 1975; Ediz and Dickerson, 33
1976; Jones and Dropkin 1976; Byrne et. al., 1977; Meon et. al., 1978; Finley, 1981; Jones, 1981; Pasha et. al., 1987).
The giant cells appeared completely enclosed by abnormal xylem elements, but the serial section study revealed that none of the giant cells was completely enveloped by the xylem. The giant cells were always connected with the phloem. The secondary phloem elements, instead of forming a complete ring, appeared diverting towards the giant cells when seen in transverse section. In this way the supply of assimilates to the giant cells was not disrupted (Hisamuddin, 1992).
Induction of giant cells in roots of olive (Olea europed) by M. javanica, and hypertrophy and hyperplasia, were common phenomena in the cortical and vascular parenchyma. (Abrantes et. al., 1992). Wyss et. al., (1992) reported invasion in Arabedopsis thaliana roots by M incognita primarily in the region of elongation, close to the meristematic zone, by destroying epidermal and subepidermal cells. Inside the root, the second-stage juveniles (Jj) oriented themselves always in the direction of root tip. At the apex of the root the juveniles turned round and migrated towards the differentiating vascular cylinder. Within the vascular cylinder, migration eventually stopped and the giant cell induction was initiated. The head of second-stage juvenile was found surrounded by multinucleate giant cells within 24 hours. Bird (1992) laid emphasis on stylet exudate proteins in establishment and maintenance of giant cells.
Host susceptibility and resistance, each results from a series of sequential events that are easily divided into pre-and post infection stages. 34
The sequential events may or may not lead to the establishment of a disease. Resistance may be defined as the absence or inhibition of disease upon challenge by a pathogenic agent. Thus, anything that prevents, retards, or restricts disease development contributes to host resistance. Aborted giant cells and dead larvae and females of M incognita were observed in roots of marigold and castor bean, but not in Chrysanthemum and tomato (Hackney and Dickerson, 1975). In castor bean roots, the infected stele harboured more males than females. Resistance manifested at the time of infection or thereafter may be constitutive or due to infection induced factors.
Weiser (1956) while experimenting with the excised roots of soybean and egg plant observed varied responses towards larvae of M hapla, some being repellent, some attractive and some neutral. He attributed these results an interplay between an attractive agent present in the living plant and a repellent associated with the chemical decay and break down of root. Studies on attractiveness root-knot nematode towards host plant roots have been carried out by Bird (1959), Loewenberg et. al, (1960) and Loewenbery and Viglierchio (1961). Malic hydrazide (multifunctional inhibitor) treatment within four days after infection by M. incognita also produced similar effects (Davide and Trinataphyllou, 1968). Terpenoid aldehydes were histochemically, demonstrated in endodermis and stele near the head of nematode. Infection induced terpenoid accumulation occurred in both susceptible and resistant plants, but accumulated in higher concentration in resistant than in the susceptible cultivars. Thus, the rate and extent of accumulation seems to be an important factor. Tomato seedlings treated with 35 morphactin (an antibiotic) that apparently does not affect nematode or at least does not affect Panagrellus redivivus but markedly delays in giant cell formation and nematode development, and induces of a tendency towards maleness in maturing larvae (Orion and Minz, 1971).
Necrosis was observed by Dean and Strubble (1953) in roots of resistant sweet potato and tomato plants invaded by root-knot nematode. Liao and Dunlap (1950) reported that root-knot nematode juveniles that penetrated tomato roots were arrested, half embedded in the tissues and suggested the presence of a chemical inhibitor. Crittenden (1958) also noted that in resistant varieties, heads of nematodes did not become surrounded by giant cells. The number and size of giant cells were reduced and their cytoplasms were sparse and not dense, resistance to root-knot nematodes in resistant cotton varieties was associated with a necrotic reaction in affected roots Brodie et. al., (1960). Nematode juveniles entered the root of resistant cotton as readily as in susceptible. In resistant variety the development of the nematode was much retarded and most of the nematodes failed to reach sexual maturity.
Minton (1962) attributed resistance of cotton roots to Meloidogyne incognita acrita to condition within the roots that prevented or delayed nematode development and not to failure of nematodes to enter the roots. Resistance in Gossypium barbadense toM incognita acrita was associated with increased root necrosis and reduced hypertrophy and hyperplasia, a corresponding reduction in tissue disorganization and gall formation, and failure of nematodes to mature. 36
Powell (1962) found no apparent differences in reactions between resistant and susceptible plants three days after inoculation. However, after 7 days, hypersensitive responses were noted in resistant tissues shown by degeneration of giant cells and collapse of cells surrounding infection loci. These reactions were progresively more apparent at 10 and 17 days after inoculation. After 17 days, the nematode bodies appeared to degenerate.
McClure et. al., (1974) reported three types of histological responses in infected resistant roots of cotton and co-related them with the degree of nematode development. Some galls were observed containing only fragments of nematodes, others contained no detectable traces of developing larvae. Formation of druses in galls, but not in healthy tissues, was noted in both resistant and susceptible plants, 20 days after inoculation. Massive invasion of roots resulted in deep longitudinal fissures of root cortex.
Failure of the giant cells to develop or to function as transfer cell results in the death of nematode and is, therefore, considered as a mechanism of resistance of the host plant. Resistant plant infected with Meloidogyne incognita normally undergoes a hypersensitive response and the affected cells turn borwn and die. However, in roots treated with cyclohexamide, the typical hypersensitive response does not occur or at least the affected cells do not turn brown and the cells remain alive. Sawhney and Webster (1975) observed browning of affected cells and dead cells, and lack of browning of living cells.
In both resistant and suceptible plants, equal number of nematode larvae penetrated but in resistant plants only a few of them developed into 37 mature females. The remaining surviving nematodes were found in various stages of development in some Solarium species (Barrons, 1939; Doncaster, 1953; Williams, 1956).
There was no difference in larval penetration of Meloidogyne incognita acrita in susceptible Cucumis melo and resistant C. ficfolius and C. mituleferus. After 26 days of infection, giant cell development v^as the same in roots associated with adult females of all the three plant species. However, in C. mituleferus immature nematodes were found associated with small giant cells (Fassuliotis, 1970). Approximately same number ofM incognita acrita larvae penetrated the resistant and susceptible roots of alfalfa plants (Reynolds et. al., 1970). in resistant roots the larval penetration decreased sharply after 3-4 days, and later on none of the nematode developed to mature stage. In susceptible roots larvae became sedentary and developed normally. Similar results were also obtained in resistant and susceptible cotton cultivars by McClure et. al., (1974), in four resistant and susceptible genotypes of corn by Windham and Williams (1994) and in Solanum melongena and S. sisymbrifolium by Fassuliotis and Dukes (1972).
According to Struble et. al, (1966) the resistance to root-knot nematode was inherited in multifactorial fashion in sweet potato. Out of 4,343 lines of sweet potato tested against M. incognita, only Nemagold variety exhibited resistance. Different types of host parasite relationships have been demonstrated: (i) trace or less amount of root galling, (ii) trace to severe root tip necrosis, (iii) few or no nematode developing into mature forms, (iv) little or no reproduction of the nematode, (v) reduced number 38 of eggs per egg mass in some cases where reproduction occurs, and (vi) few or no giant cells.
Gentile et. ai, (1962) did not find nematodes developing beyond second-stage juveniles. In addition, they found either no root tip necrosis or only traces in Tinian variety of sweet potato. In another variety Heartogold, Giamalva et. al, (1963) reported relatively little root necrosis. A new variety Bayou of cotton was found to have resistance towards root- knot nematodes that produced few egg masses per plant and the egg masses contained few eggs per egg mass. (Birchfield and Jones, 1966). Low penetration rate, slow or no development of nematode and reduction in egg mass production are some phenomena related with resistant varieties as have been described by McClure et. al, (1974) and Griffin and Elgin Jr. (1977).
Malo (1965) studied the nature of resistance of "Okinawa" and "Nemaguard" peach root stock towards Meloidogyne javanica and traced giant cell formation after 4-days in both susceptible and resistant varieties. After 10 days, accumulation of suberin like material was observed around the walls of giant cells, that walled off and isolated these structures from the rest of the cells of vascular cylinder. The walling off progressed with time causing a typical decline in giant cell formation and development. Later on giant cells became empty giving an alveolar look that had no affinity for any stain. Nematode development was abnormal and had completely stopped after advanced stages of giant cell walling off. Remaining of dead nematode larvae, and giant cells surrounded by several layers of safranin stained suberized cells, were seen in cross sections of old root tissues. Sosa-Moss 39 et. al, (1983) observed few juveniles of M. incognita in resistant cultivars of tobacco, after 7 and 14 days of inoculation. The infection site exhibited some cavities and extensive necrotic tissue after 14 days. However, after 35 days less necrotic tissue and no intact nematodes were observed. Khan and Khan (1991) observed significant difference in the penetration of M incognita race 1 and M javanica in resistant and susceptible germplasm cultivars of various vegetables.
Tacconi (1993) conducted field trials of Meloidogyne resistant and susceptible tomato in infested sandy soil in presence and absence of nematicides. The yield obtained from resistant cultivars in infested, nonfumigated soil was equivalent to those obtained from susceptible cultivars in fumigated soil. While evaluating fruit yield and quality of six local ecotypes and six exotic cultivars of okra (Abelmoschus esculentus), against Meloidogyne incognita, Hussein et. al., (1994) found Balady green and Clemson spineless to be highest yielding ecotype and cultivars, respectively. Vovlas et. al., (1994) cultured anther derived diploid line of potato from a root-knot nematode resistant parent. The resistant line, on infection with M. incognita, exhibited necrotic tissues, undersized or no giant cells, and consequently suppression of nematode development.
In two wild grasses, Aegilops and Hordeum, Balhadere (1995) studied resistance response towards M. naasi. Both exhibited hypersensitive response with a range of necrotic sites in the endodermis, thus preventing most nematodes from migrating into the stele. Creech et. al., (1995) compared rates of penetration and reproduction in resistant and susceptible 40 cotton. M. incognita race 3 exhibited delayed response in M-315 resistant genotype. In M 315, the nematode failed to establish and maintain giant cells (Jenkins et. al., 1995). Two peach root stocks, Nemared and Nemaguard were resistant against M javanica. (Philis, 1995). Out of 18 cucumber varieties tested by Sharma et. al, (1995) for their reaction to M incognita, Hoe-707 and EC-173929 were found resistant. The resistant line of Gossypium hirsutum againt. M. incognita exhibited necrosis around nematode head. In the giant cells cytoplasmic organelles were fewer as compared to susceptible plants (Tang et. al, 1995).
While screening different cultivars of pigeon pea, Bhat et. al, (1996) found all the cultivars susceptible towards M. incognita race-1. Eight genera of Leguminosae were evaluated against M chitwoodi and M. hapla. Alfalfa and red clover exhibited a response different from other genera. Afalfa survived 39 to 89% and red clover 10 to 55% at green house temperature in presence of nematode populations. All other genera survived all nematode populations (Griffm and Rumbaugh 1996).
Pedrosa et. al, (1996a) inoculated M arenaria race 1 and 2 to know resistance and susceptible responses of three genotypes of soybean, race 1 stimulated small, giant cells while race 2 induced well developed, thick w ailed, multinucleate giant cells in all the genotypes. In resistant genotype of soybean, rate of penetration and development ofM incognita race 1 and race 2 was ver>' slow as compared to susceptible genotypes (Pedrosa et. al, 1996b).
Jensen and Griffin (1997) found all auto-and allotetraploids of several 41
plant species including grasses of Triticeae to be resistant against M chitwoodi. Janssen et. al., (1997) worked on some wild genotypes of Solarium to evaluate responses of M. chitwoodi, M. fallax and M hapla. Four genotypes exhibited high resistance towards M. chitwoodi and M fallax. Two genotypes showed moderate resistance towards M fallax but no resistance towards M chitwoodi. Abdel-Momen et .al, (1998) tested Arachis breeding lines and found that several peanut lines which were resistant towards M arenaria were also resistant towards M Javanica and an undescribed Meloidogyne species.
Gupta et. al, (1995) observed that initial inoculum of 100 larvae of Meloidogyne spp. in bitter gourd 1,000 larvae in smooth gourd, ridge gourd and squash melon significantly reduced the growth parameters. Galling and nematode reproduction was directly related to initial inoculum level.
Fazal et. al, (1996) determined threshold levels of M incognita and R. reniformis on black gram. The threshold limits of M incognita and R. reniforms were 1000 J^ and 1000 immature females, respectively. After 72 and 135 days, highest population desnity (20 eggs/g of soil) of M incognita decreased growth of tomato Ehwaeti et. al, 1998). 4 and 8 eggs/ g of soil affected growth after 135 days, fresh and dry weights, oil yield, rate of photosynthesis, total chlorophyll, sugars, and phenol decreased in Ocimiim at the highest inoculum density (16000 ]Jl-5]f.g soil) of M incognita. Highest Rf (45.85) occurred in plants inoculated with 5000 J,/7-5 kg soil (Haseeb et. al, 1998). 42
CHEMICAL CONTROL:
The first use of soil nematicide. Carbon disulphide, may be traced back to a paper published in Gardener's Chronicle in 1858 (Berkeley, 1858). Carbon disulfide was reported to be "the most efficient chemical for the control of the root-knot nematodes in the field" (Bessey, 1911). After the insecticidal efficiency of carbon disulfide was established, it was also applied to control the sugarbeet nematode by Kuhn in 1981, but results were not encouraging (Thorne, 1961). Its importance as a nematicide has been demonstrated many times but it has never been used extensively (Taylor, 1959). Literature on chemical control of plant parasitic nematodes has been reviewed by several workers from time to time. Recent reviews are by Wright (1981), Hague and Gowen (1987) and Haq et. al, (1990).
During 1940s soil fumigants like DD and 1,3-D were used to kill the nematodes. Some more fumigants were also introduced later on. Recently water soluble nematicides called nonfumigants have been developed. They are classified as organophosphates and carbamates. Most of these chemicals are systemic and control many soil inhabiting or foliar feeding pests. VC- 13 (dichlofenthion), the first organophosphate, released for nematode control, was used mostly to protect ornamentals and turf grass (Christie and Perry, 1958; Good, 1963). Whether applied as a drench, spay or injected into the soil, VC-13 reduced nematode populations sufficiently to permit turf grasses to regain their former vigour and growth (Manzelli, 1955). Jenkins and Guengerich, (1959) reported thionazian as an important nematicide. 43
Another organophosphate, phenamiphos was reported to control nematodes on citrus seedlings by bare root dip (O'Bannon and Taylor, 1967) and on ornamentals as drench (Johnson, 1969a, b). The efficacy of ethoprop for the control of Meloidogyne incognita acrita was demonstrated on tobacco (Osborne et. al., (1969); and tomato (Brodie, 1971). Other organophosphates such as parathion, demeton, phorate, disulfoton, diazinon and nellite also controlled phytoparasitic nematodes.
Weiden et. al, (1965) proposed a new class of insecticidal and acricidal carbamoyloximes. Their biological activity spectrum was similar to that of certain organophosphates. In 1966 the nematicidal activity of aldicarb was reported on the tobacco cyst nematode, Heterodera tabacum (Miller, 1966), and after some time on some nematodes that infected vegetable crops (Rhoades, 1969).
Soil application of aldicarb, aldicarb sulfone, carbofuran, thionazin, phorate, fensulfothion, disulfoton, dimethoate, ethoprophos, Mocap (ethoprop), oxamyl, and thionazin + phorate were effective in reducing the nematode populations and increasing the yield of brinjal, tomato, okra, cauliflower, chillies, tobacco, ground nut, sugar beet, wheat and rice etc., (Singh and Prasad, 1973, 1974; Singh et. al., 1978; Varaprasad and Mathur, 1980; Krishnaprasad and Krishnappa, 1981; Mahajan, 1982; Reddy and Singh, 1983; Haq et. al., 1984; Hussaini, 1986; Dutt and Bhatti, 1986a, b; Sakhuja and Sethi, 1986 and Rahman, 1991).
Nematicides applied as soil drench were found to be equally effective against root-knot nematodes. Vydate (oxamyl), VC-13 and Dazomet 44 satisfactorily controlled other nematodes in general and M. incognita in particular on egg plant, okra, tomato, and chilli (Alam et. al., 1973a, b; Saxena et. al., 1974; Alam and Khan, 1983); aldicarb reduced the population of M incognita and root galling on mung bean (Vein et. al., 1977). Oxamyl- G, oxamyl-L, aldicarb, fenamiphos and fensulfothion were effective in inhibiting M. javanica population on tomato (Dabaj and Khan 1982); aldicarb treatment resulted in significantly lower root-knot indices (M incognita) on egg plants (Mahajan, 1982); aldicarb and carbofuran reduced penetration of M. incognita in roots of tomato (Dutt and Bhatti., 1986a, b); fensulfothion, dimethoate, aldicarb, and carbofuran arrested M incognita population growth and galling, and promoted tomato growth (Haq et. al. 1987). Reduction in juvenile penetration and subsequent root galling on tomato was observed with Dimecron 85%, Metasytox-R, Phorate (Thimet lOG), Carbofuran (Furadan 3G) and Phenamiphos (Nemacur 5G) (Rahman et. al., 1988; Thakar et. al., 1988). While using aldicarb, carbofuran and carbosulfan to control M. javanica on tomato, aldicarb was found most effective in controlling the nematodes and in increasing plant vigour. Phenamiphos, carbofuran and mural were more effective against Myovan/ca and M. incognita on egg plant, cucumber (Cucumis sativa L.) and okra than oxamyl, (Stephan et. al., 1988; Pravatha Reddy and Khan, 1991).
Endo-and ectoparasitic nematodes infesting cowpea were effectively controlled when fenamiphos was applied to the soil (Sethi and Meher, 1989). There was a decrease in the soil and root population of phytoparasitic nematodes viz., M. incognita, M. javanica, M. arenaria, Heterodera 45 glycines, Pratylenchus branchyurus and Helicotylenchus spp. and increase in the yield of soybean with the application of fenamiphos, carbofuran and aldicarb (Rodriguez-Kabana and Mawhinney, 1980; Novaretti et. al., 1982; Kinloch, 1983a, b). Conley et. al, (1983) reported that Temik (aldicarb) or Counter (terbufos) reduced the reproduction rate of plant parasitic nematodes associated with soybean. Contrary to this, Kinloch (1983c) and Mueller (1984) observed that, though Furadan (carbofuran) or Temik (aldicarb) increased the yield of soybean in H. glycines and M incognita infested soil, but none of them significantly reduced the nematode number.
Carbofuran, fenamiphos, bendicarb, aldicarb (Temik lOG), and ethoprop not only reduced gall number but also increased the yield of chickpea (Kaushik and Bajaj, 1981; Singh and Reddy, 1981, 1982). Combined application of Temik lOG and Brassicol also gave better results (Pandey and Singh, 1990). Temik lOG caused significant reduction inM incognita population than fenamiphos in cotton (Kirkpatrick, 1993).
Aldicarb (Reddy and Singh, 1983) controlled Me/o/c/ogy«e incognita and increased yield of french bean {Phaseolus vulgaris). Reddy, (1985 a, b) reported that fenamiphos was more effective than aldicarb and carbofuran, in controlling M incognita on pea plant. Sharma and Trivedi (1985) found fensulfothion, aldicarb or cytrolane most effective in controlling M. incognita and increasing the number of nodules on pea plant. Fensulfothion followed by aldicarb and BHC reduced root-knot index on pigeon pea, (Jaiswal et. a!., 1987). Among antagonistic plants, chicken manure, biocontrol agents and among chemical nematicides, aldicarb was found to 46 have greatest suppressive effects on gall formation and root-knot nematode population (Oduor-owino and Waudo 1995).
The effect of aldicarb, dazomet, carbofuran, thionazin, phorate, fensulfothion, disulfoton, dimethoate, mocap, terbufos, ethoprophos, isophenphos, phenamiphos and oxamyl on R. reniformis were studied on mung bean (Singh and Prasad, 1973; Patel and Thakar, 1986); soybean Birchifield and Williams, 1974; chick pea Mahapatra and Padhi, 1986); pigeon pea (Jaiswal et. al., 1987); french bean (Padhi and Mishra, 1987); garden pea (Sundaram and Velayutham, 1988). All the nematicides were effective in reducing the nematode population and increasing the yield.
When applied to the soil, oxamyl is readily taken up by the plants where it interferes in the development of the nematode (Hague and Pain 1973), but Whitehead et. al, (1973) suggested that once the nematodes enter the root they become less sensitive to nematicides applied either to the foliage or to the soil and this view was also supported by Bunt, (1975, 1977); Griffin, (1975); McLeod and Khair, (1975) and Hague, (1979).
Oxamyl when applied to the foliage of field beans and potato was found to be transported basipetally (Bunt and Noordrink, 1977; Peterson et. al., 1978; Wright et. al., 1980). Foliar application on sugar beet inhibited development of Heterodera schachtii (Griffin, 1975). It's spray on cabbage and banana roots confirmed the nematicidal activity whereas washing of the roots removed the activity (Potter and Marks 1976a, Gowen, 1977), Wright and Womack, 1981). Starr and Mai (1975) on the other hand, did not succeed in obtaining the satisfactory results after foliar application. 47
Due to oxamyl application, the feeding activity of Meloidogyne incognita was checked that hampered nematode development (Evans, 1973; McLeod and Khair, 1975; Atilano and Van Gundy, 1979). The changes in feeding behaviour, in movement of juveniles and also in development, are due to effects of oxamyl on the nervous system of the nematodes (Evans, 1973; Hague and Pain, 1973; Nelmes et. al., 1973; Le Patourel and Wright, 1974, 1976; McLeod and Khair, 1975; Lee and Atkinson, 1976; Atilano and Van Gundy, 1979; Wright e/. al., 1980; and Susan, et. al., 1982).
McLeod and Khair (1975) studied the effects of different concentrations of oxime carbamates, organophosphates, and benzimidazole on the egg hatch, juvenile viability, development of the juveniles of Meloidogyne spp. Hatching was markedly reduced by methomyl, fenamiphos and thionazin while aldicarb had little effect. Hatching was not affected by benomyl, thiobendazole. Juveniles of all the three species of Meloidogyne viz. M. hapla, M. incognita and M. javanica survived until three days when immersed in 16 to 32 ppm solutions of aldicarb, fenamiphos, ethoprophos and thionazin. Migration of juveniles varied with other nematicides, aldicarb, however, reduced the rate of migration. Aldicarb and thionazin stopped the development of M. javanica, but methomyl, ethoprophos and fenamiphos reduced the development. Nematicides that reduced the development of the nematode also prevented the normal orientation of juveniles in the roots and reduced or prevented giant cell formation. Atilano and Van Gundy (1979) also did not find any effect on tomato seedlings.
The effect of oxamyJ on the nematodes was studied in detail by Wright 48 et. al., in 1980. Oxamyl was found to be affecting neurotransmission in nematodes through inhibition of acetylcholinesterase activity. This led to change in feeding, movement and orientation behaviours. A single foliar application of oxamyl reduced invasion of cucumber seedling by M. incognita juveniles for at least 21 days. But when inside the root, their development was not checked. However, root application reduced both invasion and development significantly. Impairment of nervous system due to inhibition of actycholinesterase activity is assumed to be the mode of action of carbamate and organophosphate pesticides (Corbett, 1974; Le Patourel and Wright, 1974, 1976).
In seedlings, treated with aldicarb, methomyl, fenamiphos, ethoprophos or thionazin, McLeod and Khair (1975) did not observe giant cells but found larvae scattered in the parenchymatous tissue of outer cortex of the root.
Batterby et. al, (1977) compared accumulation and metabolism of aldicarb in two species of free living soil nematodes. Aphelenchus avenae and Panagrellus redivivus, which differ considerably in their sensitivity to aldicarb and other pesticides accumulated the nematicide to the same extent. Aldicarb uptake rate was greater in P. redivivus so also the rate of metabolism and elimination. Levels of toxic metabolites were also higher (2-3 times) in the more susceptible species A. avenae, after 24 h of incubation, than in less susceptible P. redivivus. Aldicarb at very low concentration (O.Olug cm-^) has been reported to impair sex attraction in H. schachtii (Hough and Thomson 1975), and to inhibit hatching and 49 emergence of//, cajani larvae and to inhibit development of//, rostochiensis larvae (Hague and Pain, 1970; Marban - Mendoza and Viglierchio 1980b) similar results were obtained by Greco and Thompson, 1980 by phenamiphos on H. schachtii and M. javanica. (Singh and Singh, 1993).
While working on in-vitro and in-vivo effets of aldicarb on survival and development of Heterodera schachtii, Steele and Hodges (1975) found that aqueous solutions of 5-500 )j.g/ml aldicarb inhibited hatching. Treatment of newly hatched larvae of//, schachtii with 5-500 M-g/ml aldicarb suppressed later development of larvae on sugarbeet {Beta vulgaris), a similar treatment with aldicarb sulfoxide had less effect on larval development, and aldicarb sulphone had no effect. The number of treated larvae that survived and developed were inversely proportional to concentration (0.5-5 (ig/ml) and duration (0-14 days). Nelmes et. al., (1973) detected 0.01 mg/g aldicarb sulfone in tomato roots, 16 days after dipping of roots in aldicarb solutions. Invasion of roots by M incognita was suppressed in treated plants two weeks after treatment.
The effects of aldicarb on H. schachtii in two different soils were studied by Hough et. al., (1975). They found that aldicarb effectively controlled the nematode even after 42 days of application and subsequently irrigating the soil at different intervals and at different rates. Aldicarb moved towards water flow and concentrated in the furrows where it persisted for many days and acted on the nematodes.
Hough and Thomson (1975) studied the toxic effects of sublethal concentrations of aldicarb on second-stage larvae of Meloidogyne javanica 50 and reported 100% hatch inhibition between 4.8 and 48.0ng/ml. Low concentration (0.48[ig/ml), however, stimulated the hatching of eggs. Migration of M. javanica larvae in sand column was inhibited under continuous exposure to l^ig/ml of aldicarb. Larval emergence of A/. javanica was completely inhibited at 48.0ng/ml which is in the range of concentrations that completely inhibits the egg hatch of M. arenaria (Berge and Cuany, 1972 andM incognita. (Nelmes and Keerweewan, 1970).
Hague and Pain (1973) and Zambelli and De Leonardis (1970) recovered numerous H. rostochiensis larvae from soil in rhizosphere and suggested that aldicarb between 4.8 and 4.8 jig/ml concentrations in the soil were not sufficient to prevent hatching but were enough to prevent infection. Aldicarb has been reported to inhibit undulatory movements of H. rostochiensis larvae at 1.0 |ig/ml, and to increase the toruosity of the tracks of M incognita treated with aldicarb (Nelmes, 1970). While camparing the efficacy of fumigant and non-fumigant nematicides for control of Meloidogyne chitwoodi on Solanum tuberosum. Griffin (1989) found aldicarb to be most effective when applied post plant (PP) during the nematode reproductive cycle. Nanjegowda et. o/., (1991) applied antibiotic KT 199 that markedly decreased the number of nematodes in the roots. Number of giant cells in a gall and number of nuclei in the giant cells also decreased. Giant cells contained less insoluble polysaccharides, nucleic acids and proteins. Antibiotic checked the damage, caused by M incognita, on tomato plants.
The nematicides like carbofuran, carbosulfan and phenamiphos 51 inhibited M. incognita larval penetration and their development on roots resulting in reduced gaill formation and reproduction after 50 and 60 days in bitter gourd and round melon (Pankaj and Siyanand, 1992). Fazal et. al., (1995) applied five chemicals to treat the seeds for the management of root- knot nematode. Carbofuran (40 flowable) was most effective in controlling M incognita population.
FUNGUS:
The exploitation of living organisms for reducing pest populations has been termed as biological control (Swarup and Gokte, 1986). Many natural enemies attack plant - parasitic nematodes in soil and reduce their populations. Literature pertaining to antagonistic organisms of plant nematodes has received attention of several reviewers (Mankau, 1981; Jatala, 1986; Swarup and Gokte, 1986; Khan, 1990).
It has been known for sometime that the nematode populations are naturally contained and consequently the disease incidence reduced, but the actual mechanisms involved, however, are not adequately understood. Available knowledge indicates that phytoparasitic nematodes have many natural enemies including fungi, (Kerry, 1980, Gaspard and Mankau, 1986; Rodriguez - Kabana and Morgan - Jones, 1988); bacteria (Sayre, 1980; Sturhan, 1988; Fattah et. al., 1989; Stirling et. al., 1990); and predacious nematodes (Mankau, 1980; Bilgrami and Jairajpuri, 1989). Antagonistic interactions between fungi and nematodes have been known to occur, in agricultural soils, for many years (Barron, 1977; Mankau, 1980). Fungi that 52 destroy nematodes occur in most of the soils and, undoubtedly, play an important role in regulating nematode populations. Fungi possessing the capacity of destroying or deleteriously affecting the nematodes, vary both in their biology as well as taxonomy. They consist of a great variety of forms which include endoparasitic fungi, Meria coniospora (Jansson et. ai, 1985a, b); Hirsutella rhossiliensis (Eayre et. al., 1987; Jaffe et. al., 1989); nematophagus fungi, Arthrobotrys, Dactylaria, Monascroporium, Nematoctonus (Mankau, 1961; Gaspard and Mankau, 1986); and the fungi parasitic to the eggs and cysts, Paecilomyces lilacinus (Jatala et. al, 1979), Verticillium chlamydosporium (MoTgan Jones et. ai, \9%\), Acrophialophora /M5/5p6>m (Husain, 1988).
Recent studies have indicated the existence of opportunistic soil fungi capable of colonising nematode reproductive structures and deleteriously affecting their life cycles (Rodriguez-Kabana and Morgan-Jones, 1988). Nematodes belonging to Heteroderidae {Globodera, Heterodera and Meloidogyne), at sedentary stages of their life cycles, are vulnerable to attack by these fungi either within the host plant roots or when exposed to the root surface within the soil.
Association of microflora with the eggs, females, or with the cysts of Heteroderidae was reported for the first time by Kuhn in 1877. He observed a fungus pathogenic to females of Heterodera schachtii and named it Tarichium auxiliare (Kuhn 1877, 1881), now known as Catenaria auxiliaris (Tribe, 1977).
Investigations performed by Bursnall and Tribe (1974) and Graham 53 and Stone (1975), with the cysts oi Heterodera avenae and H. schachtii, confirmed the regular occurrences of a number of fungi capable of invading them in the soil. Symptoms encountered included granular or shrivelled eggs, black eggs containing spore like bodies, and eggs with contorted juveniles (Morgan-Jones and Rodriguez Kabana, 1987). A number of mycological surveys of heteroderid nematodes conducted in the last decade (Morgan- Jones et. al., 1981, 1984a, b, Morgan-Jones and Rodriguez-Kabana, 1981; Gintis et. al., 1982, 1983; Godoy et. al., 1983, Carris et. al., 1986; Rodriguez-Kabana and Morgan-Jones, 1988; Husain, 1988) confirmed that the cysts and the eggs were frequently colonised by fungi.
Among the most frequently encountered fungi, found in significant numbers and associated with more than one species of nematode, are the species of the genera, Acremonium, Alternaria, Catenaria, Cylindrocarpon, Exophiala, Fusarium, Gliocladium, Humicola, Nematophthora, Paecilomyces, Phoma, Pythiiim, and Verticillium (Rodriguez-Kabana and Morgan-Jones, 1988).
The feasibility of utilising selected opportunistic soil fungi capable of colonising cysts and eggs for biocontrol of phytophagus nematodes is being explored (Stirling and Mankau, 1979; Crump and Kerry, 1981, 1983; Franco et. al., 1981; Kerry et. al., 1984; Rodriguez-Kabana e/. al., 1986; Husain, 1988; Khan and Husain 1988). Jatala et. al., (1979), provided Paecilomyces lilacinus to several nematologists through International Meloidogyne Project for the trial, and the data obtained revealed that this fungus adapted well in varied climatic conditions and was effective in 54 controlling root-knot nematodes under greenhouse and field conditions (Jatala, 1985, 1986; Khan and Esfahani, 1990).
Majority of the eggs of Meloidogyne incognita acrita on potato roots collected by Jatala et. ah, (1979) near Huanuco, Peru, were found infected with Paecilomyces lilacinus. When the fungus was inoculated into nematode infected potato plants, it invaded 70% of the egg masses oiMeloidogyne and 90% of the cysts of Globodera pallida, and it also penetrated mature Meloidogyne females. Under field conditions, infected potato plants, grown in plots treated with P. lilacinus, had significantly lower root gall index than those grown in plots which received the application of organic matter and the nematicides. 86% of the eggmasses obtained from plants grown in fungal treated plots were found to be infected with P. lilacinus, while 54% of the eggs were destroyed (Jatala et. al., 1980). In another experiment, on multiple and term effect of P. lilacinus in controlling M. incognita, Jatala et. al., (1981) found no difference in root gall index on potato, grown in fungus infested and non infested plots. On examination, non infested plots were found contaiminated with P. lilacinus. Therefore, any difference in colony counts of samples obtained from two plots was not noticed. They concluded that one time application of the fungus was sufficient for the establishment and reduction of nematodes.
The percentage of infested eggs increased as the time of exposure to fungus {P. lilacinus) increased, but there was no difference in egg infection in different media. Decreased hatching was observed after 25 days because of increased number of infected eggs of Globodera pallida (Franco et. 55 al., 1981).
Noe and Sasser (1984) observed that P. lilacinus increased the yield of tomato and okra and lowered the population of M ;wcog«/7o juveniles, at the mid season and at the beginning of the next season, in treated plots than in untreated plots. All the isolates of P. lilacinus, obtained from tomato, eggplant, and celer>' roots, significantly reduced egg hatching (Villanueva and Davide, 1984). The juveniles of M incognita when exposed to the fungus resulted in reduced gall formation. The local P. lilacinus isolate caused 63 to 82% reduction in gall formation when tomato roots were dipped in a fungal suspension before inoculating with 300 or 1,000 M incognita juveniles. They concluded that spore suspension of the fungus had greater control efficacy that mycelial suspension. Midha (1985) while inoculating plants with egg masses, eggs, or juveniles, found that the P. lilacinus spores had little effect where inoculum source was egg masses or eggs. This trend was evident in both, cowpea and mung. Sayre (1986) reported successful control of plant parasitic nematodes by P. lilacinus, and other fungal agents, Nematophihora gynophila and Dactylella oviparasitica, and a bacterium Pasteuria penetrans. Findings of several other studies indicated that artificial infestation of P. lilacinus in soil significantly reduced the intensity of the disease caused by root-knot nematodes (Candanedo-Lay et. al, 1982; Godoy et. al, 1983; Rodriguez-Kabana et. al, 1984; Roman and Rodriguez Marcano, 1985; Cabanillas and Barker, 1989; Dube and Smart Jr, 1987; Sharma and Trivedi, 1989).
Khan and Esfahani (1990) observed that simultaneous or sequential 56 inoculations where P. lilacinus was added prior to M. javanica were more effective in controlling the nematode on tomato than when nematodes preceded the fungus. Root galling and egg mass production was greatly recuced in the presence of the fungus. The higher doses of fungus (75gm) reduced root galling on brinjal, significantly (Sharma and Trivedi, 1989).
Cabanillas and Barker (1989) evaluated the effect of inoculum levels and time of application of P. lilacinus on the protection of tomato against M incognita. The best protection against M. incognita was attained with 10 and 20 g of fungus infested wheat kernels per microplot, which resulted in a three fold and four fold increase in tomato yield, respectively, compared to plants treated with nematode alone. Maximum protection against the pathogen was attained when P. lilacinus was delivered into the soil 10 days prior, to and at the time of planting. Percentage of P. lilacinus infected egg masses was greatest in plots treated at mid-season or at midseason plus an early application, compared with plots treated with fungus 10 days before planting and/or at the planting time. During inoculum level studies it was found that 1 or 2g per pot of P. lilacinus significantly reduced the damage caused by R. reniformis (Khan and Husain, 1986).
Cabanillas, et. al., (1988) studied the histological interaction of Paecilomyces lilacinus with Meloidogyne incognita on the excised roots of tomato. They observed no giant cell in tomato roots inoculated with nematode eggs infected with P. lilacinus. Few to no galls and no giant cell formation were found in roots dipped in spore suspension of P. lilacinus and inoculated with M incognita. Numerous large galls and giant cells were 57 present in roots inoculated with M incognita only. P. lilacinus colonised the surface of epidermal cells as well as the internal cells of epidermis and cortex (Dunn et al, 1982).
Cameiro and Gomes (1993) prepared twelve isolates of P. lilacinus and P. fumosoroseus, and all of them were found effective in destroying eggs of M javanica. Lin et. al., (1993) used P. lilacinus to control root- knot nematode. Meloidogyne incognita. In laboratory tests, P. lilacinus parasitized 54% eggs. In field trials, the fungus parasitized 40% eggs when the fungus was applied at transplanting time at the rate of 8 g per experimental plot. Amoncho and Sasser (1995) in greenhouse trials found that P. lilacinus colonized rice and okra but it suppressed root galling on okra and not on rice. According to Mittal et. al., (1995) M. incognita, causing disease on Solanum melongena, Lycopersicon esculentum and Cicer arietinum, was suppressed more in presence of P. lilacinus and chitin than using them alone. P. lilacinus suppressed 83% M. javanica reproduction (Ibrahim, 1994) and use of inoculum pellets of P. lilacinus in soil showed greater suppression in root-knot index as compared to its application as soil drench or seed pelleting (Shahzad, et. al., 1996).
Oduor-owino and Waudo (1996) reported effects of five fungal isolates on egg hatching and parasitism on eggs, juveniles, and females of M arenaria, M. incognita and M javanica. Paecilomyces lilacinus and Fusahum oxysporum had the greatest suppressive effects on hatching. They also parasitised more than 70% of eggs and females. However, F. oxysporum parasitised less than 20% eggs. The controls showed highest proportion of 58 egg hatch, and F. oxysporum treated eggs exhibited least level of parasitism. The fungus caused infection in eggs of M javanica, M. incognita and M. arenaria.
Fazal et. al, (1996) reported reduced nematode population and increased plant growth of black gram with P. lilacinus alone and in combination with furadan. The best combination of these components, with regard to reduction in gall number, nematode population, and increased plant growth of infected plants was at 2g P. lilacinus combined with l.Og a.i. furadan.
Vyas et al., 1996 while testing efficacy of fenamiphos, carbofuran and Paecilomyces lilacinus alone and in combination for management of M incognita spp. in chick pea, found fenamiphos to be most effective followed by P. lilacinus. P. lilacinus alone or in combination with oil cakes, leaf extracts of castor and neem, and carbofuran reduced nematode (M incognita) populations on okra and tuberose {Polianthia tuberosa) (Nagesh et. al., 1997 and Rao et. al., 1997). Cardona and Leguezmon (1997) found P. lilacinus causing 94% infection in females and almost 94% infection in eggs and 100% infection in juveniles of Meloidogyne species.
To protect chick pea fromM incognita, Bhat et. al., (1998) tried P. lilacinus and oil cakes of neem and mustard. They observed a significant increase in plant growth, nodulation, and suppression in reproductive factor of M incognita as compared to plants inoculated with nematode alone. Large number of egg masses were found infested with the fungus. Reduction in 59 gall index and increase in yield on application of P. lilacinus on tomato in M javanica infested field was reported by Zaki (1998).
Khan and Williams (1998) reported significant reduction in M. javanica populations in tomato by P. lilacinus. TEM and SEM studies revealed that the fungus penetrated the female body through the body wall rather than only through natural openings. Juveniles within eggs were also found infected. SECTION-I
EXPERIMENT 1.
Effect of Paecilomyces Ulacinus on the histopathology of Meloidogyne incognita infected Momordica charantiaplants.
The fungus Paecilomyces Ulacinus (Thorn) Samson has been reported as a potential biological control agent for root-knot nematodes and other plant parasitic nematodes. (Jatala et. al., 1979; Franco et. al, 1981; Jatala, 1982, 1986; Adiko, 1984; Cardona and Leguezamon, 1997 and Khan and Williams, 1998). P. Ulacinus is a common soil hyphomycete closely related to Penicillium (Samson, 1975). The sedentary stages of root-knot and cyst nematodes are most vulnerable to P. Ulacinus. The fungus is capable of colonizing nematode reproductive structures thus causing destruction of females, cysts and eggs. (Franco et. al, 1981; Jatala, 1982, 1986; Gintis, et. al, 1983 and Cardona and Leguezamon, 1997).
Paecilomyces Ulacinus parasitises eggs of Meloidogyne spp. and Glohoderapallida {Dunn et. al, 1982; Jatala, 1986). The fungus exhibits chitinase activity when grown in chitin agar plates (Gintis, et. al, 1983). The fungus shows a wide antimicrobial activity against some other fungi, yeast, and Gram positive bacteria due to the production of peptidal antibiotic (Isogai et. al, 1980, 1981). Paecilomyces Ulacinus increased the yield of 61
tomato and okra and lowered the population of M />icog«i7a juveniles, at the mid and at the begining of the next season in treated plots than in untreated plots (Noe and Sasser, 1984). M />icog«/7o juveniles when exposed to the fungus resulted in reduced gall formation and egg mass production. (Villanueva and Davide, 1984; and Khan and Esfahani, 1990).
Although the consistent association of P. lilacinus with eggs of Meloidogyne spp. and its ability to penetrate both eggs and females is well documented, (Franco, et. al, 1981; Jatala, 1982, 1986; Adiko, 1984; Freire and Bridge, 1985), the exact mode of its parasitism is unknown. Root galling and giant cell formation were absent in tomato roots inoculated with fungus infected eggs of A/, incognita. P. lilacinus colonized surface of epidermal cells as well as the internal cells of cortex of tomato roots (Cabanillas ef. al., 1988). The effects of fungus on the M incognita parasitising the roots oiMomordica charantia has not yet been reported. The objectives of this study were to (i) examine the effect of P. lilacinus on M. incognita infected plant tissues, (ii) know the effect of P. lilacinus on nematode development, (iii) determine the effect of P. lilacinus on eggs and egg masses, (iv) examine the effect of P. lilacinus on the giant cells, and (v) to evaluate the efficacy of P. lilacinus in controlling the disease by applying at varying time intervals. The experiment was designed as per the following treatment scheme.
(1). T, — uninoculated control plants — 0 nematode. 62
(2). T^ — inoculated with 1000 J2 of M/wcogm/a.
(3). T3 — inoculated with 1000 J^ and treated with fungus one week before inoculation.
(4). T^ — inoculated with 1000 J2, and treated with fungus simultaneously.
(5). Tj — inoculated with 1000 J2, and treated with fungus one week after inoculation.
(6). T^ — inoculated with 1000 J2, and treated with fungus 2 weeks after inoculation.
(7). T., — inoculated with 1000 J2, and treated with fungus 3 weeks after incoulation.
Each treat ment was replicated five times for growth parameters and for histological studies, and the pots were arranged in a randomized complete block design. The data for different parameters were determined and statistically analysed for significance of variance at P< 0.05 and P< 0.01 levels by ANOVA.
MATERIALS AND METHODS
SELECTION OF TEST PATHOGEN, TEST FUNGUS, AND TEST PLANT: The root-knot nematode, Meloidogyne incognita (Kofoid and white. 63
1919, Chitwood, 1949) was selected as test pathogen. The fungus Paecilomyces lilacinus was selected as test biocontrol agent &nd Momordica charantia as test plant.
COLLECTION OF INOCULUM OFMELOIDOGYNEINCOGNITA: Meloidogyne incognita infected egg plant roots were collected from vegetable crop fields. The root-knot nematode species was identified on the basis of characteristic perineal pattern and differential host test. The pure cultures of M incognita was raised and maintained on egg plants using single egg mass. The egg mass obtained from egg plant roots infected with M. incognita, was surface sterilized with chlorox (Sodium hypochlorite 0.5%) (NaOCl) solution for five minutes. It was washed thrice with sterilized distilled water and allowed to hatch at 27°C in an incubator.
Egg plant seedlings raised in 25 cm diameter pots containing autoclaved soil were inoculated with second stage juveniles. In order to maintain sufficient inoculum throughout the course of investigations new eggplants were inoculated with at least 15 egg masses obtained from pure culture.
Isolation and Preparation of Nematode Inoculum: Large number of egg masses from heavily infested egg plant roots were hand picked with the help of sterilized forceps from previously maintained pure culture of M incognita. The egg masses were washed with distilled water and placed in sieve containing crossed layer of tissue paper. 64
The sieve was kept in petri dish containing water just touching its lower surface. A series of such assemblies were kept to obtain required number of second stage juveniles for inoculation. The hatched out juveniles were collected from petri dishes after every 24 hours and transferred to a beaker. Fresh water was added to petri dishes to repeat the process. The water suspension of the nematodes was gently stirred to obtain homogenized distribution of nematodes. From the nematode suspension five counts were made with the help of counting dish (Southey, 1986) under stereoscopic microscope and an average of these counts was taken to determine the density of nematodes per unit volume of the suspension. The volume of nematode suspension was so adjusted that each ml contained 1000±2 juveniles.
Preparation and Sterilization of Soil Mixture: Sandy loam soil was sieved through 16 mesh sieve to remove stones and debris etc. The soil was then thoroughly mixed with organic manure in the ratio of 2:1. Earthen pots of 15 cm diameter were filled with this soil and organic manure mixture of 1kg per pot. In each pot the soil was moistened with water. The pots were autoclaved to sterilize at 20 lb pressure for 20 minutes. Sterilized pots were allowed to lose their heat at room temperature before use for the experiments.
RAISING AND MAINTENANCE OF TEST PLANTS: Axenization of Seeds: The seeds of Momordica charantia (L.) obtained from National Seeds 65
Corporation, New Delhi were axenized with NaOCl (Koenning and Barker, 1985). About 200 seeds were poured into sterilized beaker. The beaker was filled with 1:1 mixture of 95% ethanol and 5.25% NaOCl. The mixture was stirred gently and allowed to soak for about 8-10 minutes. The mixture of alcohol and NaOCl was drained out and the seeds were rinsed with sterile distilled water.
Germination of Seeds:
About 10-20 axenized seeds were placed in a moist sterilized filter paper kept in a sterilized petri dish. The seeds were allowed to germinate. The germinated seeds were transferred to 15cm diameter clay pots filled with steam sterilized soil.
Inoculation with Nematode: When the seedlings became one week old, holes of 5-7cm depth around the plants within a radius of 2 cm were made. Through these holes 1000 second-stage juveniles (J^) were introduced with the help of sterilized pipette.
Preparation of Fungal inoculum: The culture of Paecilomyces lilacinus used in the experiments was obtained from International Potato Centre, Lima, Peru. Richards liquid medium (Riker and Riker, 1936) was used for its mass production. The mycelia were blended to make mycelial suspension for soil application. The Richard's liquid medium consists of the following components: 66
Potassium nitrate KNO, lO.OOg
Potassium dihydrogen phosphate KHjPO^ 5.00g
Magnesium sulphate Mg SO^.THp 2.50g
Ferric chloride FeClj 0.02g
Sucrose Cj.H^jO^ 50.00g
Distilled water 1000ml
The medium was prepared, filtered through muslin cloth, and sterilized in an autoclave at 15 lb for 15 minutes in 250ml corning flasks, each containing 100ml of liquid medium. The medium was inoculated with small amount of fungus maintained on PDA slants with the help of sterilized inoculating needle under aseptic conditions in aseptic chamber. These incoulated flasks were incubated at 28.5°C for about 15 days to allow rapid fungal growth for experimental purposes.
Preparation of Fungal Inoculum and its Inoculation: After enmassing oi Paecilomyces lilacinus, lOOg of mycelia were blended in 1000 ml of distilled water in warring blender such that 10ml of suspension consisted of one gram mycelia.
Inoculation:
The fungus P. lilacinus was incorporated into the soil around the root 67
zone of bitter gourd at the rate of 1 .Og per pot. Holes of 5-7cm depth around the plants within a radius of 2cm were made. Through these holes l.Og per 10ml of distilled water fungal mycelia were pipetted. The holes were plugged with sterilized soil. To maintain soil moisture, the pots were regularly watered.
HISTOPATHOLOGICAL STUDIES Inoculated seedlings were uprooted carefully after 45 days. The roots were washed gently and thoroughly to remove all soil particles adhering on them. Galled roots were cut into 1cm long pieces and processed for histopathological studies. The infested roots were processed as follows.
(1). Fixation. (2). Dehydration, (3). Infiltration, (4) Embedding. (5). Sectioning (Ribbon Mounting), (6). Staining.
Fixation: The pieces of galled roots and healthy roots were immersed in a fixative of formalin-aceto-alcohol (F.A.A), prepared by mixing 90ml of 50% ethanol, 5ml of glacial acetic acid, and 5ml of 37% formaldehyde (Johansen , 1940). Depending upon thickness of galled tissues it was kept in the fixative for a minimum of 24 hours.
Dehydration: The galled and healthy root tissues were dehydrated through tertiar>' - but\l -alcohol (TBA)schedule as given by Johansen (1940) (Table 1). 68
Infiltration: After dehydration by alcohols, paraffin was introduced into the root tissue. After step 8 of table (1) the tissue was transferred to a mixture of 100% TBA and paraffin oil in the ratio of 1:1. It was kept for at least one hour or more in this mixture. Another container half filled with melted wax was allowed to solidify. The tissue was placed on the wax and then filled with TBA and paraffin oil mixture.lt was kept in an oven at about 65°C. After three hours the mixture was poured off and replaced with pure melted wax. The container was kept at the same temperature for about three hours This step was repeated twice.
Embedding: For embedding, molds were prepared from thick paper by folding it. The molds were coated with glycerol from inside. Some melted paraffin wax was poured into the molds. As the wax started solidifying on the bottom of the mold, the root pieces were placed on it with the help of heated forceps. More melted wax was added when the wax around the tissue solidified.The molds were kept for some time untill the wax completely solidified . After hardening, the whole block was cut into small pieces according to the position of root pieces.
Sectioning: The small blocks of wax enclosing root tissue were trimmed to remove extra VN ax. The wax blocks were mounted on wooden blocks and then fixed 69
in rotary microtome. With the help of knife, 10-12|im thick sections were obtained in the form of a ribbon.
Ribbon Mounting: Ribbons were cut into smaller lengths according to the length of the slides. Surface of slide was coated with Haupt's adhesive (Johansen, 1940). The ribbon was placed on the slide and flooded with freshly prepared 2-3% formalin solution. These slides were kept in an oven at 40°C for 6-8 h, and then stored in slide boxes.
Staining: The sections were stained with safranin and fast green after removing paraffin by the method described by Sass (1951) (Table -2). The slides were taken out from xylene. Mounting medium was applied on the surface of the slide before evaporation of xylene. Cover-slip was lowered gradually over the slide.
Finished slides were left at room temperature for at least 24 h and then kept in an incubator at 60°C. The slides were examined under light microscope. The necessary photographs were taken .
GROWTH PARAMETERS:
Forty five days after inoculation the plants were terminated and following parameters were taken into account, for describing the results, (i) length (root and shoot), (ii) fresh and dry wieght of root and shoot, (iii) 70
number of branches, (iv) number of flowers, (v) leaf area, (vi) number of egg masses per plant, (vii) number of galls per plant, and (viii) size of gall.
OBSERVATIONS ROOT AND SHOOT LENGTH: Maximum length of both roots and shoots was observed in control plants. Among all the treatments, T^ plants were shortest that were inoculated with nematode and were not integrated with Paecilomyces lilacinus. The root and the shoot were significantly (P<0.01) shorter than Tj plants. Reduction in root length was more (43.42%) than in shoot length (32.39%). Root and shoot lengths of Tj plants as compared to T, plants were slightly but not significantly more. In T^ plants nonsignificant reduction in root and shoot lengths was observed. The root and shoot lengths of Tj, T^ and T, plants decreased, where nematode inoculated plants were treated with P. lilacinus after one, two and three weeks of inoculation, respectively. Reduction in length was more in T,than in T^ and Tj plants. Thus T, plants were shortest among nematode inoculated and P. lilacinus treated plants.
The root and shoot lengths of T^ and T^ plants significantly (P< 0.01) increased when compared with only nematode inoculated (T^) plants. In the treatments where nematode inoculated plants were supplied with P. lilacinus after two and three weeks of inoculation, the plants lengths were higher than T^ plant length. Increase in length, however, was non significant. 71
FRESH WEIGHT: The fresh weight of roots and shoots of Momordica charantia decreased significantly (P<0.01) in 1^ plants as a result of infection of M incognita, when compared with iminoculated control. Reduction was higher in roots (46.02%) than in shoots (38.33%). In comparison to T^ plants a significant (P< 0.01) increase in weights was observed in Tj plants in which P. lilacinus was applied one week before nematode inoculation. The fresh weights of control Tj and Tj plants were more or less the same. As compared to nematode inoculated plants (Tj) only, a significant increase was also observed in T^ plants in which P. lilacinus was applied simultaneously with nematode inoculation. The fresh weight of T^ plants was lower than (Tj) control plants, however, the differences were non significant.
Fresh weights of roots and shoots of T^ plants in which P. lilacinus was applied one week after nematode inoculation, decreased significantly (P < 0.01), when compared with control. A gradual decrease in fresh weights with an increase in time interval of P. lilacinus application, after nematode inoculation, was encountered when the data were compared with control plants. Minimum reduction in P. lilacinus treated plants was found in T, plants where P. lilacinus was applied after three weeks after nematode inoculation. In comparison to only nematode inoculated plant (T^), an increase in weight was found in all the P. lilacinus treated plants. Gain in weight was maximum in T3 plants that received P. lilacinus one week before 72
inoculation and minimum in T, plants in which it was given after three weeks of inoculation. Increase in weight was also significant in T^ plants where P. lilacinus was applied simultaneously, and non significant in Tj, T^ and T^ plants (Table 3).
DRY WEIGHTS OF ROOTS AND SHOOTS: Dry weights of roots and shoots significantly (P< 0.01) decreased in Tj plants inoculated with M. incognita, when compared with uninoculated control (Tj) plants. Reduction was more (43.95%) in roots than in shoots (37.11%). Dry weight of T, and T^ plants increased significantly (P< 0.01) in comparison to only nematode inoculated {T^ plants. There were no significant differences between T, and T^ plants and between Tj and T^ plants. The dr>' weights of T, plants were similar to Tj plants. The dry weights of T^ plants were lower than T, plants, the differences, were non significant.
On comparing with control plants the dry weights of roots as well as of shoots of TJ, T^ an T^ plants were significantly lower. The data show a gradual decrease in weights with an increase in time interval of application of P. lilacinus. Among P. lilacinus treated plants T^ plants exhibited maximum reduction.
The root and shoot weights of T3 plants and T^ plants were much higher as compared to that of T.^ plants. Increase in weight was significantly (P < 0.01) high in both the treatments. An increase in weight was also observed 73
in T5, Tg and T, plants when their weights were compared with T^ plants. The differences were non significant. The differences were more in Tj plants followed by T^ and T, plants (Table 3).
NUMBER OF BRANCHES AND FLOWERS: Meloidogyne incognita infected plants which were not treated with F. lilacinus produced fewer branches and lower number of flowers. The number of branches and flowers significantly (P < 0.01) increased in T, and T^ plants that were treated with P. lilacinus before one week and together with nematode inoculum, as compared to T^ plants. The number of branches and flowers decreased in T^, T^ and T^ plants when compared with (T,) control plants. Their number was significantly lower. Although, number of branches and flowers of T^, T^ and T^ plants increased when compared with T^ plants, the increase was non significant (Table 3).
LEAF AREA: The leaves of control plants were large sized and also of T, and T^ plants. The leaf area of T, plants and T, plants was almost the same. The leaf area of T_, plants was slightly lower than T, and T, plants. The leaves of T^ plants were small and their leaf area was significantly lower than T, plants. The leaf size of T^, T^ and T^ plants also decreased when compared with control. But, when compared with T^ plants the leaf areas of T^, T^and T^ plant were found to be larger (Table 3). 74
GALLS: Root-knot nematode caused severe galling in Implants. The number of galls per plant was very high (183.40). Their number reduced considerably in Tj plants where P. lilacinus was applied one week before nematode inoculation, in which few galls per plant were observed. The number of galls per plant was also considerably lower on T^ plants that were integrated with P. lilacinus at the time of nematode inoculation. The gall number was considerably and significantly high on Tj, T^ and T, plants. T, plants produced maximum number of galls followed by T^ and the Tj, among M. incognita inoculated and P. lilacinus treated plants (Table 3).
SIZE OF GALL: M. incognita infection produced large sized galls on the roots of Momordica charantia. The average size of gall was (7.40mm^). Integration of P. lilacinus resulted in significant reduction of gall size. The highest reduction in gall size was noticed on T3 plants treated with P. lilacinus one week before nematode inoculation. The galls on T^, Tj, T^ and T^ were smaller than those of T^ plants. Among P. lilacinus treated plants largest galls were observed on T^ plants. The size of gall increased on the plants integrated with P. lilacinus one week after to three weeks after nematode inoculation.
NUMBER OF EGG MASSES PER PLANT: On TJ plants large number of egg masses were found to be associated with the galls. M incognita produced large number of egg masses (83.6) 75
on M. charantia roots. Integration of P. lilacinus resulted in reduction in egg mass production. The number of egg mass per plant was significantly low (12.11) on T3 and (14.60) T^ plants in which P. lilacinus was given one week before inoculation and simultaneously with nematodes, respectively.
The number of egg mass per plant increased on T^ plants in which P. lilacinus was applied after one week of inoculation. The number was further higher in T, followed by T^ and T, plants. Highest number of egg masses was found in T, plants among P. lilacinus treated plants (Table 3).
HISTOPATHOLOGY:
NORMAL ROOT OF MOMORDICA CHARANTIA:
The root system of Momordica charantia normally consists of short tap root and large number of lateral branches. These secondary lateral branches further devide profusely resulting in a highly branched root system. Histologically the primary roots of M. charantia consist of unisereate epidermis, multilayered parenchymatous cortex, and stele. The stele in primary root may be diarch, triarch but generally tetrarch, displaying a t>'pical dicotyledonous pattern. Xylem and phloem are radially arranged alternating with one another. There is also a small pith consisting of parenchyma cells at the centre of the four xylem arches (Fig. 1).
Secondary growth initiation in the M charantia roots takes place when the vascular cambium starts functioning. The vascular cambium appears 76
first on inner edges of the primary phloem and then on the outer side of the primary xylem strands. Soon the two cambia are joined so that a continuous, but a wavy ring is formed. Once the cambial ring is complete, it begins to form secondary vascular tissues; secondary xylem centripetally and secondar>' phloem centrifugally. As a result of differentiation of xylem and phloem in the two direction the wavy ring of cambium assumes a circular outline in transverse section. The secondary vascular tissues so formed are in the form of two continuous cylinders, phloem being an outer and xylem an inner cylinder. As the secondary growth progresses, the pith and protoxylem are crushed. Root-knot nematode inoculated plants receiving no nematode control treatment produced large sized galls. Fully mature females were found feeding on giant cells. Near the giant cells more amount of abnormal xylem and phloem was observed (Fig. 2). All the mature females were found associated with egg masses. Anatomical details of the galled roots of the plants which were given Paecilomyces lilacimts treatment, one week before nematode inoculation, revealed that the fungus entered the root tissue and grew successfully. Thehyphae and conidiophores bearing chains of conidia could be seen in the normal vessel elements of the xylem (Fig. 3, 4). In these plants, the galls were much smaller as compared to the galls observed on T, plants. The giant cells though smaller but resembled with those of T^ plants. In the vicinity of the giant cells abnormal xylem and abnormal phloem was present. The amount of abnormal vascular elements was less. The fungal 77
hyphae destroyed eggs and egg masses (Fig. 5) and also entered into the body of the females (Fig. 5 ). Aroimd the nematode body fungal growth was abundant (Fig. 5). The root surfaces also exhibited profused growth of the fungus (Fig. 6, 7).
The simultaneous application of root-knot nematode and P. lilacinus not only destroyed eggs and egg masses (Fig. 8) but also entered the internal tissues of the root, either intercellularly or intracellularly as is evident from the transverse section of vessel elements (Fig. 9). The egg masses were destroyed by the fungus. The growth of fungus was profound inside the egg masses. There was no change in the size of giant cells and amount of abnormal vascular elements as compared to untreated plants.
In the plants where P. lilacinus treatment was given one week after nematode inoculation, the fungal hyphae were observed inside the giant cells (Fig. 10). The fungal growth was profuse around the body of the developing nematode (Fig. 11). In the normal tissues, the fungus spread both inter- and intracellularly as is evident from the figure. (Fig. 12).
Similarly, in the plants where P. lilacinus was applied two weeks after nematode inoculation, the fungal hyphae destroyed the eggs masses (Fig. 13). The fungus was also observed in the abnormal xylem elements developed in response of root-knot nematode infection (Fig. 14). The amount of abnormal xylem was more as compared to T, and T treatments.
^ —- r I ( Ace. N
'^^^.y; m 78
In the plants, in which P. lilacinus was given after three weeks of nematode inoculation, the giant cells around the nematode head were observed (Fig. 15). The fungal hyphae surrounded the females (Fig. 16). The vessel elements were seen having hyphae and conidia in the normal and galled roots of this treatment also (Fig. 17). The size of giant cells as well as amount of abnormal xylem and phloem increased.
In all the treatments, in which P. lilacinus was applied, gall formation had occurred but the size of the galls was invariably smaller than untreated plants. The galled regions comprised of abnormal xylem and phloem in addition to hyperplastied parenchyma and giant cells. The giant cells helped in the development of the juveniles to adult feamles. P. lilacinus consistantly damaged and destroyed eggs and egg masses of M. incognita. It also colonized root surface and surface of the mature female. Occassionaly, hyphae were also observed inside the giant cells and inside the body of the nematode.
DISCUSSION Healthy plants ofMomordica charantia attain a length of about one and a half meter in the pots. The shoot length is usually more than one meter and the root length is about 30cm. The plant length was drastically reduced w hen infected with Meloidogyne incognita. The effect was more on roots than on shoots. After application of Paecilomyces lilacinus an improvement in plant length was observed. As the data indicated the shoot as well as the 79
root lengths reduced greatly in presence of M incognita only. Stunting of the plant shoot as well as root as a result of root-knot nematode infection has been reported by several workers. (Oteifa, 1952; Barker and Olthof, 1976; Olthof and Potter, 1972; Rodriguez - Kabana and Williams; 1981).
In the treatment (T3) where Paecilomyces lilacinus was applied to the soil just before the transplanting of germinating seedlings and one week before nematode inoculation the plants exhibited a significant increase in both root and shoot lengths as compared with nematode inoculated plants. The lengths of root as well as shoot were almost equal to that of control plants. From these observations it is evident that P. lilacinus contributed in controlling infection and maintaining the plant health. Cabanillas and Barker (1989) has also reported three to four times increase in growth of M. incognita infected tomato where the fungus was applied 10 days before plantation. The advantage of applying P. lilacinus before seedling transplantation is that the fungus being saprophytic is established in the soil (Brown and Smith, 1957). Since it is also endophytic (Cabanillas, et. al. 1988) so the hyphae penetrate in the growing primary and secondary roots. Being parasite of mature M incognita females it would destroy them (Jatala, et. al., 1979; Freire and Bridge, 1985) and also parasitize the egg masses (Jatala, et. al., 1979; Jatala, 1982, 1986; Morgan Jones and Rodriguez- Kabana, 1984; Freire and Bridge 1985).
The T^ plants in which P. lilacinus was introduced at the time of 80
nematode inoculation the plant length reduced non significantly in comparison to control, and increased significantly when compared with T^ plants, inoculated with only nematode. From this observation it is evident that the nematode had established successfully before the fungus could penetrate into the roots. This resulted in lowering of gall formation that caused slight reduction in root and shoot lengths.
Significant reduction in root and shoot lengths of Tj plants in which P. lilacinus was given after one week of inoculation suggests that the fungus did not destroy M incognita females considerably. This caused higher infection of roots leading to lesser plant growth. The plants of T^ and T^ treatments exhibited higher reduction in root and shoot length. In these treatments the fungus was applied two weeks and three weeks after inoculation. Because of late application of P. lilacinus probabily it could not reach all the mature female and all the egg masses that resulted in still higher infection. The consequence of higher infection even in presence of fungus was that the plant length enormously reduced.
Meloidogyne incognita not only caused heavy reduction in plant length but also in fresh and dry weights of Momordica charantia in the treatment 1^, as is evident from the table - 3. Higher reduction in weights of roots than on shoots may be due to the reduction in root length and reduction in number of secondary branches. Although fibrous roots are formed in higher numbers but these are very thin and short, therefore, contribute very 81
little in root weight. Reduction in root and shoot weights was mainly due to dwarfing and stunting that might be caused by several mechanisms. Nematodes remove plant nutrients, alter nutrient flow patterns in the plant tissue, and retard root growth, all of which may be attributed in suppressing plant yield (Hussey, 1985; Barker and Olthof, 1976). Oteifa, 1952 observed retarded shoot growth and nutritional deficiency symptoms in foliage of root-knot nematode infected plants. Root branching and linear root extension are checked by Meloidogyne infection (Hunter 1958). According to him reduced root surface area caused poor nutrient uptake that ultimately resulted in suppressed growth of infected plants.
A significant increase in fresh and dry weights of roots and shoots of Tj plants, in comparison to T^ plants, may be attributed to the biocontrol agent P. lilacinus. Before planting the seedlings P. lilacinus established in the soil, entered into the roots and worked to minimize the infection. The fungus contributed in controlling secondary infection by destroying the maturing females and the egg masses. Similar findings were also reported by (Cabanillas and Barker 1989) in tomato.
A similar trend was also observed in T^ plants but the gain in weights was lower than Tj plants. Application of P. lilacinus simultaneously with nematode inoculation was not as much effective as when applied one week before inoculation. Gain in weights was higher in T3 plants than T^ plants although the differences were non significant with control Tj plants. 82
Simultaneous application of P. lilacinus at the time of M. incognita inoculation was not found as effective as 10 days before inoculation in tomato plant by Cabanillas and Barker (1989). Integration of F. lilacinus after one week or more weeks of inoculation caused significant reductions in fresh and dry weights of roots and shoots. The results indicate that as the time of fungus integration is increased reduction in plant growth is also increased as is evident from higher reduction in weight in plants treated with fungus after three weeks of inoculation as compared to that of one week after inoculation. The time of application of fungus seems to be important in controlling root-knot disease. These results are in accordance with Cabanillas and Barker (1989) who worked on tomato.
Root-knot nematode infection causes reduction in number of branches in addition to shortening of plant height (Hunter. 1958). The same was observed in the case of Momordica charantia. Paecilomyces lilacinus not only increased root and shoot weights but also enhanced branch number as is evident from the data. Branching in T, plants was at par with control plants. Although in T^ plants number of branches decreased but were not significantly different from the Tj control plants. This shows that P. lilacinus when applied in soil before transplanting the seedlings or at the time of nematode inoculation, increased plant length, plant weight and also number of branches. It means P. lilacinus effectively controls nematode infection and improves the health of the plants. 83
Application of P. lilacimts after nematode inoculation, did not improve plant growth satisfactorily as can be observed from the data. M incognita infected plants even in presence of P. lilacinus remained shorter and had few number of branches. It shows that P. lilacinus before or at the time of inoculation is more effective as compared to that where it was applied one, two or three weeks after inoculation.
In M. incognita infected plants, flowering was affected considerably. It may be because of lesser amount of transport of nutrients from root to shoot. Root-knot nematode infection destroys many soybean cultivars causing yield reductions (Kinloch, 1980). Suppressed yield of susceptible tobacco in presence of M incognita and M. javanica has been reported by Arens and Rich (1981). In T, and T^ treatments P. lilacinus not only improved plant growth but also enhanced flowering.
In other treatments where M. incognita infected plants were treated w ith P. lilacinus, two and three weeks after inoculation, did not exhibit any significant increase in flower number as compared to control, and T,, T^ plants. This may be attributed to P. lilacinus activity, where it could not control root-knot nemtaode disease effectively. Integration of P. lilacinus before inoculation or at the time of inoculation controls disease to a greater extent. Since in other treatments disease is not controlled and, therefore, flowering remains affected alongwith the plant growth.
The leaf size in M incognita infected plants is reduced. Leaf area is 84
probably reduced because of low up-take of minerals and water and their transport to above ground parts. Reduction in shoot weight is caused by shortening of plant but more importantly by small sized leaves. The leaves of T3 and T^ plants were larger as compared to Tj and T^ plants. Increase in their leaf area may be credited to P. lilacinus which controlled nematode infection to a significant level. If leaf area is increased, more food will be synthesized that will increase plant growth.
In other treatments reduced leaf area indicated higher damage in plant growth, when compared with control. In these treatments P. lilacinus could not control the disease effectively that resulted in damage to the leaves in general and plant as a whole.
Underground symptoms of M. incognita infected plants are the presence of galls on their roots (Molliard, 1900; Christie, 1936 and Mueller, 1984) M charantia also produced galls on infecting with M incognita. Integration of P. lilacinus before nematode inoculation or simultaneously caused reduction in gall formation.
When the fungus was applied any time after one week of inoculation, no significant change in gall number was observed as is evident from T,, T^ and T, plants. This indicated that the nematode had established in the roots before the establishment of the fungus. The data show a gradual increase in size of the galls from Tj to T,. This indicates that the efficacy of the fungus against M incognita diminished as the time of application increased. M. 85
incognita infected plants, not treated with biocontrol agent P. lilacinus, exhibited large sized galls. The gall size was reduced to greater extent in T^ and T^ plants. This indicates that either few juveniles had entered the roots or their development had slowed down or secondary infection had completely checked. Most of the egg masses collected from these treatments were destroyed by the fungus. Even mature females had been parasitized by P. lilacinus and had killed them. Increase in number of egg masses on T^, T^ and T, plants indicated that P. lilacinus had not parasitized them. Low infection of P. lilacinus on egg masses was probably because of late establishment of fungus. On T.^ plants egg mass number was quite high showing an ineffectiveness of the fungus at the later stage.
As soon as the development of M incognita female is completed it lays eggs in the form of egg masses. A large number of egg masses were observed on T2 plants infected with M. incognita and not integrated with P. lilacinus. On T, and T^ plants, no or very few viable eggs could be observed as is evident from the data. It was probably due to destruction of eggs by the already established mycelia oi P. lilacinus. The fungus disintegrated egg protoplasm and consumed it. Thus the egg masses produced by the surviving females were destroyed by P. lilacinus. This totally checked secondary infection cuased by second generation juveniles. In other treatments, the nematodes had established successfully before P. lilacinus integration, therefore, egg mass production was high. The fungus was able to reach few 86
nematodes and their egg masses.
The fungus Paecilomyces lilacinus shows diverse modes of habits.
Basically it is a saprophyte (Domsch et. al, 1980) and can easily be grown on artificial culture media. At one time it acts as an epiphyte and grows on the surface of plant roots (Cabanillas et. al. 1988). At other times it grows inside the root tissue and behaves as an endophyte and does not cause any damage to the plant. Still at other times it parasitizes eggs and egg masses of Melotdogyne species and destroy them. Beause of this lastly stated beahviour, P. lilacinus has been used, by several workers, as a biocontrol agent against root-knot and other nematodes (Jatala et. al. 1979 and Morgan
- Jones and Rodriguez - Kabana, 1984).
P. lilacinus was encountered frequently in and around normal and abnormal xylem. In our opinion, vessels and vessel elements provide sufficient space for its development and also provide an uninterrupted passage to grow inside the plant tissues. Though, Cabanillas et. al. (1988) mentioned its presence in the cortex but did not disclose its mode of penetration and growth inside the tissues. We consider that it grows and develop inside the root tissue inter- and intracellularly. Whether, the fungus is beneficial or not to the plant, but, in our opinion, it is not harmful. In all the sections studied, the fungus was not found damaging the plant tissues even when it was in abundance. Further it did not affect the giant cells in which its occurrence was noticed. In all the treatments it had been regularly observed 87
that P. lilacimts damaged the eggs and the egg masses. Various workers have reported egg detroying activity of this fungus (Jatala et.al. 1979, Godoy et al., 1983, Jatala, 1986, Jatala 1985). It has also been reported that the fungus can destory neither the juveniles nor the adult females (Jatala, 1986). The eggs, however, seem to be the most preferred target, for obtaining the nourishment, by the fungus. Contrary to this Cardona and Leguizmen (1997) reported 94% infection in Meloidogyne spp. by P. lilacinus strain 9201. Khan and Williams (1998) found P. lilacinus entering into the body of the females through natural opening. They did not mention whether the fungus damaged the female or not although it damaged the eggs while inside the egg masses. Small sized giant cells and small amount of abnormalities in the xylem and phloem indicated that the nematode activity and development was influenced by the presence of the nematode. Larger giant cells and more quantity of abnormal tissues showed that the nematodes entered earlier affected by the fungus. On the basis of these observations, we conclude that the fugus can not check root-knot nematode infection at primary level when the plants are attacked by the juveniles. However, it can invariably check secondary infection because it destroys eggs as and when these are deposited. As far as time of application of P. lilacinus is concerned, from our studies, it can be suggested that incorporation of fungus one week before and at the time of nematode inoculation is more effective in controlling the root-knot disease, as compared to later intervals of incorporation. EXPERIMENT 2.
Effect of a systemic nematicide (aldicarb) on the growth, and histopathology of roots, of Momordica charantia infected with Meloidogyne incognita.
Nematicides produce different effects on the root-knot nematode development and gall formation. Aldicarb a systemic nematicide acts by contact action, disorganizing feeding behaviour of cyst and root-knot on nematodes that are living freely in the soil as well as those parasitising the roots (Wright, 1981). Aldicarb has been proved as an excellent nematicide in a wide variety of soil types round the world, against several nematode species on sugarcane (Maris, 1975). At normal dosage rates (2-12 kg a.i. ha-1), its controlling effect lasts for about six weeks.
Studies were conducted to elucidate the effect of aldicarb on the growth of plant and the development of gall formed by Meloidogyne incognita. In this study, fresh and dry weights of roots and shoots of inoculated plants were compared with control plants, and the plants treated with aldicarb at different time intervals. Leaf size, flower number, and branch number were also estimated to compare any effect of M incognita in absence and presence of aldicarb. Also, number of galls, size of galls and number of egg masses per plant were worked out. Histological changes occurring in the galled tissue of roots oi Momordica charantia inoculated 89 with M incognita were compared with inoculated and aldicarb treated plant.
MATERIALS AND METHODS PREPARATION OF TEST PLANTS: Surface sterilized (Koenning and Barker, 1985) seeds oiMomordica charaniia (National Seeds Corporation, New Delhi) were soaked in water and allowed to germinate on whatman filter paper placed in 10cm diameter petri dishes. The seeds were allowed to germinate for three days. The germinated seeds were transferred to 30 cm diameter clay pots filled with steam sterilized soil in the ratio of 7 clay: 3 sand: 1 manure.
Inoculation of Meloidogyne incognita: The inoculation of Momordica charaniia plants was done with 1000 J, ofM />7co^n/7a as described in Experiment No. 1.
Preparation of Nematicide Solution: The crystals of aldicarb 2 - methyl - 2-(methylthio propion aldehyde- 0-(methyl carbomyl) oxime) available under trade name Temik was obtained from Union Carbide. In order to obtain 1,000 ppm, Igm aldicarb was dissolved in 1,000 ml of distilled water. The stock solution was kept as such and used for different treatments.
Application of aldicarb: 1ml of 1,000 ppm of aldicarb was incorporated in rhizosphere zone of bitter gourd (M. charaniia) with the help of 1ml pipette. Aldicarb was 90 introduced through holes which were later plugged with sterilized soil. The plants were kept as such and watered regularly. The experiment was designed as per following scheme.
(1). T, - uninoculated control - 00 nematode .
(2). Tj - Inoculated with 1,000 Jj of M incognita .
(3). T- - Inoculatedwithl,000J2andtreated with aldicarb simultaneously.
(4). Tj - Inoculated with 1,000 J^ and treated with aldicarb after one week of inoculation.
(5). Tj - Inoculated with 1,000 Jj and treated with aldicarb after two weeks of inoculation.
(6). T^ - Inoculated with 1,000 J^ and treated with aldicarb after three weeks of inoculation.
(7). T^ - Inoculated with 1,000 Jj and treated with aldicarb after four weeks of inoculation.
Each treatment was replicated five times for growth parameters and for histological studies, and the pots were arranged in a randomized complete block design. The data for different parameters were determined and statistically analysed by ANOVA.
GROWTH PARAMETERS: Length (root and shoot) was taken were measured by meter scale and 91 recorded. Fresh weight of roots and shoots were taken and both root and shoots were then placed in bamboo paper envelopes. The envelopes, containing material, were kept in an oven for 48 hours at SO^C and then weighed to obtain their dry weight.
NUMBER AND SIZE OF GALL: The number of galls was counted by visual observation. The size of medium sized galls was obtained by measuring maximum length and width in (millimeters) on a meter scale.
NUMBER OF EGG MASSES: The number of egg masses per plant was counted by staining roots with phloxin B. An aqueous solution of phloxin B (0.15g per liter of water) was prepared. Galled roots were placed in this solution for 15-20 minutes and then rinsed in tap water. Red stained egg masses, were counted.
HISTOPATHOLOGICAL CHANGES: A few galls selected from each treatment were fixed in F.A.A. (Formalin aceto alcohol) and dehydrated through T.B.A. schedule. After dehydration the galls were infiltrated with paraffin wax and then embedded in it. The embedded galls were trimmed to small blocks and then fixed on wooden blocks. 10-12pm thick sections were obtained with the help of rotary microtome. The sections were mounted on slides and kept in an incubator at 40°C for few hours. (Johansen, 1940). The sections were stained with safranin and fast green as described by Sass (1951). Anatomical details were 92 observ ed under light microscope, and photographs of relevant sections were taken.
OBSERVATIONS LENGTH OF ROOT AND SHOOT: Meloidogyne incognita caused stunting of Momordica charantia. Both root and shoot lengths of infected plants (T^) significantly (P<0.01) decreased when compared with uninoculated and untreated control (T,) plants. The reduction in length reached to an extent of 40.84% and 35.51% in roots and shoots, respectively. Reductions in length were also observed in nematode inoculated and aldicarb treated plants. The extent of damage caused by nematode was different at different time intervals of aldicarb application. In comparison to control, decrease in length of (T,) plants was very small (4.56°o) in root and (3.18%) in shoot, and nonsignificant. Aldicarb was applied at the time of nematode inoculation in (T,) plants. Reductions in length of roots and shoots of T^ plants were 12.46% and 14.51% and of T^ plants were 21.72% and 25.66%. In T^ plants aldicarb was applied after one week and in T^ plants after two weeks of inoculation (Table 4).
Application of aldicarb after three weeks of nematode inoculation provided less protection to T^ plants against M incognita (Table-4). The reductions in root and shoot lengths were 32.39% and 28.76%, respectively and were significant when compared w ith control. In T^ plants, where the nematicide was given after four weeks of inoculation, heavy reduction was observed. The reductions, in comparison to control, were significant and 93 accounted 41.68% and 34.03% in roots and shoots, respectively. On comparing nematode inoculated and aldicarb treated plants with those of nematode inoculated and untreated (T^) plants, increase in length of both roots and shoots was observed. Increase with a significant difference was observed in Tjand T^ plants in which nematicide was supplied simultaneously and after one week of inoculation. The increasing trend decreased with the increase in time interval. A non-significant increase was found even after four weeks of inoculation (Table 4).
FRESH WEIGHT OF ROOT AND SHOOT: Meloidogyne incognita caused significant reduction in fresh weight of roots and shoots. The reduction was maximum (39.05%) in roots and 34.52%) in shoots in T^ plants having M incognita only. Aldicarb treatment resulted in an improvement in the fresh weight of roots and shoots. Simultaneous application of aldicarb at the time of nematode inoculation resulted in significant increase (P<0.01) in T, plants as compared to T^ plants. However, in comparison to control, the T, plants exhibited reduction which was non significant. On comparing with control, it was observed that the fresh weight of roots and shoots decreased gradually from T^ to T.^. The reductions in comparison to control, were significant in all these treatments. The data reveal that in aldicarb treated plants the fresh weight was minimum in T^ plants followed by T^, Tj and T^ plants.
Increase in fresh weight of T^ and T^ plants was significant when 94 compared with T^ plants. The weight of T^ and T., plants also increased in comparison to T^ but the increase was non significant (Table 4).
DRY WEIGHTS OF ROOT AND SHOOT: Dry weights of roots and shoots significantly reduced in all the treatments as compared to control plants. However, the over all response of the plants towards aldicarb application was beneficial. The reduction in dry weight of roots and shoots of T^ plants was highest i.e. 37.17% and 36.60% respectively, as compared to (Tj) control plants. Simultaneous application of aldicarb withM incognita inoculation increased dry weight of roots and shoots of T3 plants in comparison to T^ plants. In comparison to the control plants the weights of T^ plants were found to be lower, however, their difference was non significant.
Dry weights of roots and shoots of T^ plants, in which aldicarb was applied after one week of inoculation, significantly decreased when compared with control. But in comparison to T^ plants, the weight of the plants exhibited significant increase. In comparison to control, the dry weight of the plants of the treatments Tj, T^, and T^ significantly decreased. Among nematode inoculated and aldicarb treated plants maximum reduction was found in T., plant, 32.69% and 34.64 in root and shoot respectively, followed by Tg and T, plants. On comparing the values of dry weight of T^, T^ and T., plants with that of T^ plants it was observed that all these treatments gained weight. The increase in weight was significant in all the treatments except in T, plants (Table 4). 95
NUMBER OF BRANCHES: Branching in shoot ofMomordica charantia was found to be adversely affected by Meloidogyne incognita infection. As compared with T, plants number of branches were few in T^ plants and were significantly low (P<0.01) exhibiting 30.40% reduction. Treatment of nematode infected plant with aldicarb restored branch number to certain extent.
In comparison to control, there was no significant difference in branch number of T, plants where aldicarb tieatment was given simultaneously with nematode inoculation. After one, two, three and four weeks of inoculation, a significant reduction in branch number was found. While comparing with T., plants i.e. nematode inoculated and untreated plants, a significant increase in number of branching was observed in T, plants. In rest of the treatments the number of branches increased but it was statistically non significant (Table 4).
LEAF AREA: Size of the leaf lamina ofMomordica charantia decreased as a result of root-knot nematode infection. After treating nematode infected plants with aldicarb an increase in leaf size was observed. Maximum reduction (36.88%) in leaf area was found in T^ plants. The leaf size of T, plants was almost the same as that of control plants. There was no significant difference in the leaf area of T, plants and T, plants. In comparison to control the leaf area of T^ to T^ plants was significantly lower. When compared with T^ plants. 96 leaf area was found to be significantly higher in Tj, T^ and T, plants. Among aldicarb treated plants, maximum increase in leaf area was observed in Tj plants and maximum reduction in T, plants (Table 4).
NUMBER OF FLOWERS: Flowering is adversely affected when the plants are infected with Meloidogyne incognita. In Momordica charantia, the number of flowers per plant reduced by 31.80% in presence of infection caused by M incognita. Supplying aldicarb at the time of nematode inoculation resulted in an increase in flower number on T^ plants. Flower number was significantly (P<0.01) higher in T3 plants than T^ plants. There was no significant difference between Tj and T^ plants.
The number of flowers was significantly (P<0.01) higher on T^ plants in which the plants were treated after one week of inoculation, as compared with, Tj plants. Increased number of flowerswa s also found on T,, T^ and T, plants when compared with T^ plants, however, the increase was non significant. In comparison to Tj control plants, significantly less flowers were observed on T^, T,, T^ and T^ plants. Most affected, among aldicarb treated plants belonged to treatment T^ which produced very few flowers showing 30.38% loss over control (Table 4).
NUMBER OF GALLS PER PLANT : Severe galling was observed on roots oiMomordica charantia plants infected with Meloidogyne incognita. The application of aldicarb resulted 97 in lowering the gall production and improving the plant growth. Highest number of galls (409.00) was observed on T^ plants that were inoculated with M. incognita and not treated with aldicarb.
The gall number decreased to its lowest value (75.00) in Tj plants among aldicarb treated plants accounting 81.66% reduction in galling. In these plants, aldicarb was supplied at the time of nematode inoculation. Reduction in gall number to 120 on Implants was also significant where the plants were treated with aldicarb after one week of inoculation. Gall formation was also affected on other plants as a result of aldicarb treatment. The reduction was non significant on these Tj, T^ and T, plants (Table 4).
SIZE OF GALL : Size of the gall, as measured in length x width, ranged upto 7.40mm^ on nematode inoculated and untreated T^ plants. Aldicarb effectively decreased gall size at different time intervals. The gall size significantly (P<0.01) reduced on plants receiving simultaneous application of aldicarb in (T3), plants. The gall size on T3 plants reduced to about 45.94%. The plants treated after one week of inoculation also produced small sized galls. The gall size of T^ plants was also significantly low as compared to T^ plants. The galls were also smaller on T,, T^ and T^ plants. Their size was not significantly smaller. Largest galls among aldicarb treated plants were observed on T^ plants (Table 4). 98
NUMBER OF EGG MASSES PER PLANT : The root-knots, fonned on T^ plants inoculated with Meloidogyne incognita and not treated with aldicarb, were associated with largest number of egg masses. On the Tj plants the number of egg masses reduced to very low value showing reduction of about 41.62%. These plants were treated with aldicarb at the time of nematode inoculation. Significant reduction in egg mass number was also observed on T^, Tj and T^ plants that were treated one week, two weeks and three weeks after inoculation, when compared with T^ plants. The number of egg masses was higher on T, plants and was significantly different with T^ plants (Table 4).
HISTOPATHOLOGY Meloidogyne incognita infected (T^) plants, not treated with aldicarb, produced large sized galls. As the anatomy of galled regions reveals, the adult females along with their egg masses were frequently present in almost all parts of the root (Fig. 18). Near the nematode head, discrete giant cells surrounded by abnormal xylem and abnormal phloem were observed (Fig. 19). The giant cells were prominent and possessed dense and gran.iular cytoplasm containing multinucleolate nuclei. The giant cells were connected with phloem strands (Fig. 20).
The galls on Tj plants were much smaller. When longitudinal and transverse sections of galls of T, plants were examined, it was found that most of the nematodes were in developing stages (Fig. 21). Adjacent to the 99 nematode head prominent but smaller giant cells were present. Around the giant cells, small amount of abnormal xylem and phloem was observed (Fig. 21). Most parts of the roots exhibited normal anatomy and did not harbour any nematode (Fig. 22). Such roots, when seen in transverse section or longitudinal section, appeared to have normal xylem and phloem elements (Figs. 22, 23).
In T^ plants, in which aldicarb treatment was given after one week of inoculation, the galls were larger than that of T3 plants. From the sections of the galls, it was found that some of the nematodes developed into adult females and most of them did not develop to maturity (Fig. 24). The giant cells were quite prominent near the developing females and abnormal xylem and phloem occurred near the giant cells. Most part of the roots was free of nematodes and had no abnormal tissues (Fig. 25).
The number of females and the number of giant cells increased in the roots of (Tj) plants that received aldicarb treatment after two weeks of inoculation. The nematodes developed into mature forms and induced hypertrophy and hyperplasia. Most parts of the roots were occupied by the adult females, giant cell complexes and abnormal vascular tissues (Fig. 26 ).
Mature females with their egg masses were observed in the roots of the (Tg) plants which were treated with aldicarb after three weeks of nematode inoculation. The nematodes occurred in the cortex, conjunctive tissue and xylem arches. The nematodes were associated with giant cells 100 abnormal xylem and phloem elements (Fig. 27).
In T, plants the galled roots exhibited distortion in orientation of all kinds of tissues when seen in transverse section (Fig. 28) and longitudinal section (Fig. 29). There were multiple giant cell complexes and several fully developed females in all kinds of tissues of the galled roots. All the giant cell complexes were surrounded by abnormal vascular tissues (Fig. 30). In these roots the giant cells were large and contained large nuclei and dense cytoplasm (Fig. 31).
DISCUSSION The control plants of Momordica charantia were about more than one meter tall when grown in 15cm diameter clay pots. Meloidogyne incognita infected plants (T^) could be distinguished from the control plants on the basis of their lengths. The infected plants, not recieving any treatment, were stunted with their root lengths had severely retarded. Mere above ground symptoms are sufficient to identify root-knot nematode infection.
Aldicarb a non fumigant systemic nematicide adversely affects M. incognita at all the stages of its development (Mc Leod and Khair, 1975). Aldicarb when applied simultaneously with nematode inoculation caused significant gain in plant length. The root as well as shoot lengths of T3 plants reached upto the level of control plants. From this observation it can be inferred that aldicarb effectively controlled the root-knot nematode infection. Our findings are in accordance with the earlier investigations 101 carried out by several workers, (Mahajan, 1982; Novaretti et. ai, 1982; Kinloch, 1983a, b; Haq e/. al., 1987; Rodriguez-Kabana and Mawhinney, 1989). Application of aldicarb inM incognita infected plants, after one week of inoculation, resulted in significant increase over T^ plants. This enhancement was not sufficient to make the plant equivalent to control plants. This shows that aldicarb acts on developing nematodes but is not as much effective as to kill all the nematodes. Since it reduced infection so the plant height increased but as disease was not controlled completely, therefore, the plants remained shorter than the control as well as the Tj plants. Thakar et al. (1988) reported increased plant vigour by aldicarb, carbofuran, and carbosulfan of M incognita infected tomato plants.
The efficacy of aldicarb to check the disease decreased as the time of application increased beyond one week of inoculation. The root and shoot lengths of T5 and T^ plants were almost similar. The data further showed the root and shoot lengths of these plants were significantly smaller than the control. Although their roots and shoots were longer than T^plants but the differences were non significant. From these findings it may be concluded that aldicarb influenced on nematode development to certain extent but reduced plant length indicated that the disease was not checked completely.
The T^ plants were shortest among aldicarb treated plants. These were significantly smaller than the control plants and were taller than T^ plants. These observations suggest that effect of aldicarb was not as immense as in 102 the treatments T, and T^. The T, plants being taller than T^ plants denote that the disease was checked only to a little extent.
Significantly higher reductions were observed in fresh weights and dry weights of M. charantia plants infected with M incognita. Roots exhibited higher reductions as compared to shoots. More reduction in root weights may be attributed to their shorter lengths and to lower number of primary, secondary and tertiary branches. The plant length is affected as a result of abnormalities occurring in galled tissues leading to low up take of nutrients and water from the soil and their transport to the shoot. The dry weights and fresh weights of roots and shoots of M incognita infected Momordica charantia plants (T3) enormously increased when the plants were treated with aldicarb at the time of nematode incoulation. This increase in weight of T, plants was due to the activity of aldicarb that either killed the nematode, or checked their movement in the soil, or slowed down their rate of penetration as well as their development. This is in consistence with the previous studies carried out by Nelmes and Keerwan, (1970). Similarly, reduced penetration of M. incognita in tomato in presence of aldicarb and carbofuran has been reported by (Dutt and Bhatti, 1986a, b). Aldicarb prevented larval developement in tomato roots (Mc Leod and Khair, 1975), effectively controlled root-knot disease on tomato and increased the plant vigour (Thakar et. al., 1988). Similar observations were also reported by Pankaj and Siyanand, (1992), while working on round melon and bitter gourd.
The dry weights and fresh weights of roots and shoots of T^ plants 103 were although lower than the control and T, plants, but higher than T^ plants. Significantly higher values of weights of T^ plants than T^ plants might be due to reduced infection. The dry weights of roots and shoots of T, plants were lower than T^ plants but higher than T^ plants. The weights of these plants reduced because aldicarb was applied at later stage and caused little harmful impact onM incognita. Because of which a considerable number of nematodes entered the plants and affected plant growth.
In the treatments T^ and T^ dry and fresh weights of both roots and shoots were much lower than control plants. The figures of dry weights and fresh weights of T^ and T^ were non significantly different from T^ plants. From these observations it may be concluded that the nematode entered into the roots and developed normally. Aldicarb did not affect the nematodes that had entered into the roots. This process confirms thefindingsof whitehead et. al, (1973) and Bunt (1975).
Momordica charantia when grown in pots usually produces 4-10 branches. In presence of infection of M incognita the number of branches reduced as is evident from T^ plants. Treatment of M incognita infected plants with aldicarb improved plant growth in terms of increased number of branches. Application of aldicarb simultaneously with nematode inoculation increased branch number significantly as compared to T^ plants. Among other aldicarb treated plants, T, plants produced highest number of branches. From these observations, it may be deduced that simultaneous 104 application of aldicarb not only checked nematode infection but also improved plant growth. The plants infected with M incognita and treated with aldicarb after one week of inoculation, exhibited increase in the number of branches as compared to T^ plants but not to the extent of T3 and control plants. It shows that the efficacy of aldicarb against M incognita diminishes with the increase in time of aldicarb application from the day of inoculation.
Treatment of M incognita infected plants with aldicarb after two weeks of inoculation (T^) was not as much efficient as that of simultaneous treatment. The T^ plants exhibited branching pattern more or less similar to T, plants. Significantly higher number of branches in Tj plants, as compared to J2 plants, showed better performance of M incognita inoculated plants treated with aldicarb. However, application of aldicarb after two weeks of inoculation decreased the efficacy of the nematicide to check the infection.
In T^ and T^ plants, number of branches were much lower as compared to other aldicarb treated plants, but were higher than T^ plants in which nematicide was not applied. These findings suggest that application of nematicide after three and four weeks of nematode inoculation is beneficial for the plant to certain extent. That is why the plant exhibited better growth than Tj plants.
In control plants, the leaves are healthy and broad with large sufrace area. The leaves of T^ plants infected with M incognita remained small. Formation of smaller leaves might be attributed to low up-take and transport 105 of minerals and water from the soil to the leaves which hampered normal photosynthesis (Oteifa, 1952, Hussey, 1985). Reduction in leaf area is thus responsible in low production of photosynthates. Since a large amount of photosynthates from the leaves are diverted to the giant cells, therefore, overall plant growth is retarded.
Momordica charantia plants inoculated with M incognita and simultaneously treated with aldicarb produced large sized leaves, similar to the leaves of control plants. This indicates that aldicarb effectively checked nematode infection either by killing the nematode or by slowing down the movement of juveniles, or by reducing rate of penetration of juveniles into the roots, or by hampering the development of the nematode (Nelmes and Keerweewan, 1970; McLeod and Khair, 1975; Dutt and Bhatti, 1986a, b). In T^ plants the leaves were a bit smaller than Tj plants but significantly larger than Tj plants. It means that application of aldicarb after one week of inoculation also checks infection to a greater extent.
The leaf area of Tj plants was lower than T3 and T^ plants and higher than Tj plants. It is evident from this observation that even after two weeks of nematode inoculation aldicarb application is effective against infection. The leaves of T^ and T, plants were smaller than other aldicarb treated plants. Their size was larger, but not significantly, as compared to Tj plants. After three and four weeks of inoculation the treatment of aldicarb enhances plant growth by checking the disease to a little extent. 106
Momordica charantia plants infected with M incognita produced smaller number of flowers as compared to control. On nematode infection, flowering is adversesely affected alongwith the plant growth. Lowering in flower number leads to lower yield of fruits. Kinloch, (1982) reported yeild reduction in soybean cultivars by Meloidogyne spp.
In nematode infected plants, application of aldicarb at the time of nematode incoulation enhances flower number which appears to be directly proportional to the plant growth. In the treatment T^, increase in number of flower indicates normal supply of nutrients from the roots as well as photosynthates from leaves to the flowers. This is possible only when the plants are free of nematodes. This is the indication of the absence of nematode infection due to aldicarb treatment. In T^ plants the flower number was lower than Tj plants and much higher than T^ plants. From this finding it can be assumed that even after one week of incoulation, aldicarb is effective against the nematodes. Reduction in flower number in Tj plants as compared to T^ plants shows that treatment of aldicarb after two weeks of inoculation is beneficial to the plant but not as efficiently as in simultaneous treatment or after one week of incoulation.
Integration of aldicarb after three and four weeks of inoculation did not increase flowering. On T^ and T^ plants, few flowers were observed their numbers were not significantly different from each other. It indicates that aldicarb is ineffective in controlling nematode infection after three and four weeks of nematode inoculation. 107
Meloidogym incognita infected plants of M charantia produced large number of galls on their roots. The main roots as well as secondar> and tertiary root branches produced galls. Simultaneous treatment of infected plants with aldicarb reduced gall number to considerably . In these (T,) plants, most of the roots were gall free and healthy. New branches arising from primary or secondary roots were free of galls. Lowering in gall number on T, plants as a result of aldicarb treatment might be due to low penetration of juveniles into the roots. A few nematodes that managed to enter the roots were responsible for the formation of galls. These results are in accordance with the findings of Mahajan, (1982) who reported significant reduction in root-knot index on egg plant seedlings treated with aldicarb. In another experiment aldicarb was found effective in arresting population growth of M. incognita, reduction in gall number, and improvement in growth of tomato (Haq et. al., 1987).
In other treatments, the data reveal a gradual increase in gall number from Tj to T., which is corresponding to the time interval of aldicarb treatment. In T^ and T, plants the number of galls ranged between one hundred to two hundred. This number is significantly much lower than T^ plants indicating a harmful effect of aldicarb on M incognita. On T^ and T, the gall number increased to a sufficiently higher level. From this observation it can be deduced that enough nematodes penetrated the roots prior to aldicarb application. Moreover after three and four weeks of inoculation aldicarb could not kill the developing nematodes inside the root tissues. 108
The Tj plants having nematode infection but not treated with nematicide (alidcarb) produced large sized galls. Since the plants were not treated with aldicarb, therefore, the young roots were also attacked by the second generation of the larvae causing secondary infection. Thus, large sized galls were produced because of primary and secondary root-knot nematode infections. The gall size was drastically reduced on the roots of T, plants that were treated simultaneously with nematicide. Small sized galls indicate fewer nematodes in the galls. Steele and Hodges, 1975 reported suppressed development of H. schachtii females in sugar beet roots by aldciarb. Similarly McLeod and Khair, 1975; reported only young females in tomato seedling treated with organophosphate and oximecarbamate and benzimidazole nematicides. Atilano and Van Gundy, 1979, reported reduced size of adult females of M. javanica by oxamyl on tomato roots. Siyanand et. al., (1986) and Pankaj and Siyanand, (1992), reported reduction in gall size by nematcides. The galls on T, plants were almost of equal size probably because, probably same number of nematodes entered the gall tisssues.
On T^ and T, plants, the galls were larger but not as large as on T^ plants and also not as small as on T, plants. These plants produced large galls because of higher rate of penetration of the nematodes into roots. Among nematode infected and aldicarb treated plants, the size of galls was large in T, plants followed by T^ plants. These treatments received nematicides at later stage, therefore, the nematodes got sufficient time to enter into the roots and then develop successfully. 109
As the nematodes mature they lay eggs in the form of egg masses. The galls of T^ plants were found to be associated with a large number of eggmasses. On (Tj) plants the number of egg masses reduced drastically which can be correlated with the number of nematodes entered and matured into saccate females in the roots. Definitely few nematodes will produce few egg masses. Wright and Womack, (1980) reported similar results and found no viable egg production by oxamyl treated M ;>2cog«/7o juveniles which developed into females. The number of egg masses was also significantly low on the roots of T^ and Tj plants in which aldicarb was given after one and two weeks of inoculation. Low egg mass number on these T^ and Tj plants can be attributed to the activity of aldicarb that killed the nematode and also slowed down development of surviving nematodes in the galled tissues. Egg mass number on T^ and T^ plants being considerably high indicated a successful host parasite realtionship. In these treatments the nematodes had entered the roots and developed much before the application of aldicarb.
Meloidogyne incognita changes root anatomy, at infection site, to such an extent that it becomes difficult to demarcate normal xylem and phloem elements in longitudinal and transverse sections. If the infection is severe then the distortion in the tissues is severe. Our studies confirmed that as a result of root-knot nematode infection all kinds of tissues at the site of infection become disorganized. 110
On Tj plants, simultaneous appliction of aldicarb decreased gall index. Number of infection sites also decreased in the roots of Tj plants. Although, the nematodes induced giant cell formation, but the size of the giant cells was small. Moreover, abnormalities in the tissue adjacent to the giant cells were not conspicuous. Lowering in nematode number inside the galled tissue, reduction in giant cell size, and decrease in amount of abnormal vascular tissues might be attributed to deleterious effects of alidcarb on M. inocgnita. Being a systemic nematicide, aldicarb probably affected nervous system of the nematode. As has been reported by Evans (1973), it inhibits acts on acetylcholine esterase activity and leads to impairment in nervous system. The nematode movement is slowed down in presence of aldicarb (Evans, 1973, McLeod and Khair, 1975). Slow pace of juveniles prevented them from entering into the root tissues and the plants were safeguarded from the nematodes. Some nematodes that entered the root caused giant cell formation. Since their rate of development was also slow, therefore, the potential of causing many and large sized giant cells decreased. Abnormal xylem and phloem formation, near the giant cells, delayed probably because of weaker capability of nematodes to cause proper infection. These findings showed that aldicarb also affected those nematode that had entered the roots. The nematodes under the influence of aldicarb are unable to develop normally, and to induce proper giant cells.
Application of nematicide, after one week of inoculation, probably Ill affected nematodes even when they were inside the root tissues. This was assumed after examining the sections of galled roots of T^ plants. The number of nematodes that entered roots was high but most of them were found in developing stages. A few nematodes had reached maturity. The nematodes inside the roots produced discrete giant cells and abnormal xylem and phloem. The delayed development of the nematode may be ascribed to detrimental activity of aldicarb towards the nematodes. Increased amount of abnormal xylem and phloem and formation of prominent giant cells were probably because of the penetration of the juveniles successfully before the application of aldicarb. The nematodes inside the root tissue are less affcected by aldicarb as compared to those that remained outside the root tissue (Whitehead et. al., 1973).
In the treatments Tj, T^ and T,, the nematodes observed in the galled roots were all in their mature forms. Most of them were associated with egg masses. Around the nematode heads, presence of large sized giant cells indicated that they had entered and developed successfully and were not affected«by aldicarb. Abnormal xylem was in plenty around the giant cells. The vascular tissue also consisted of abnormal phloem.
In all the treatments from T3 to T^, it was found that eggs did not hatch, probably because of residual effects of aldicarb. The freshly hatched juveniles when come in contact with alidcarb, could not enter the roots through new locations. Instead, they all are killed before penetrating into 112 the roots. In all aldicarb treated plants, the secondary infection was prevented. McLeod and Khair (1975) also found aldicarb inhibiting egg hatching. Thus, aldicarb is most effective when applied simultaneously with the nematodes. At this level, primary inoculum is destroyed. Applicatin of aldicarb after nematode inoculation may retard nematode development if applied after short duration of nematode inoculation. The secondary infection is completely checked as a result of the presence of residues of aldicarb in the soil that affect second stage juveniles. SECTION - II
EXPERIMENT 3.
The effect of different inoculum levels of Meloidogyne incognita on Momordica charantia
Low or high population densities of Meloidogyne incognita produce different effects on plants. At low inoculum levels, sometimes the plant growth is stimulated, but at other times it is suppressed. Wallace (1971) found an increased plant growth at lower population densities and decreased growth at higher densities. Dropkin (1954) inoculated tomato roots with a single juvenile of Meloidogyne incognita acrita and measured the size of the resulted gall. He hypothesised that each individual nematode produced a finite response on the root tissue and by measuring the gall size, number of nematodes in that gall might be predicted. In heavily infested roots it was very difficult to follow the hypothesis.
The following work was carried out to determine the effects of different inoculum levels of M incognita on (i) plant growth, (ii) number and size of galls, (iii) number of egg masses per plant, and (iv) anatomical changes in the galled roots. The results were compared with the earlier findings reported for different plants by different workers. Histopathological studies of the galls, produced as a result of different inoculum levels, were also carried out. Such studies have not been carried out so far. For this 114 purpose (1) size of the mature females, (2) size of the giant cells, (3) nature of the giant cell cytoplasm, nuclei and nucleoli and (4) abnormalities in xylem and phloem were studied.
MATERIALS AND METHODS
PREPARATION OF TEST PLANTS: Surface sterilised seeds of Momordica charantia (National Seeds Corporation, New Delhi) were allowed to germinate in 10cm diameter petridishes. Three germinating seeds were transferred to 30cm diameter clay pots filled with steam sterilised soil (clay: sand: manure: 7:3:1). After one week the seedlings were thinned to one seedling per pot.
INOCULATION: Egg masses oiMeloidogyne incognita were collected from roots of egg plants and were maintained in glass house for pure culturing. The egg masses were incubated in coarse sieves (3 cm diameter) fitted with double layered tissue paper and placed on a Baerman funnel containing sufficient water. After 72h the juveniles were collected and stored in a beaker at 7°C. The number of juveniles were counted and standardised. Different inoculum levels, comprising of 05, 50, 500, and 5,000 juveniles per 10 ml of water were used to inoculate one week old seedlings. Nematode suspension containing desired inoculum levels was added in the pots with the help of sterilised pipette. Within a radius of 2cm from the plant, 3-4cm deep holes were made through which suspension was introduced. The holes were plugged 115 with soil soon after inoculation. Each treatment was replicated five times and pots were arranged in randomised complete block design. Uninoculated plants served as control. The plants were watered regularly when required and were harvested 45 days after inoculation. The data for different parameters was collected and statistically analysed by ANOVA.
PLANT GROWTH: After termination of the experiment, lengths, fresh weights and dry weights of roots and shoots of inoculated and uninoculated plants were determined. Root and shoot length was measured with the help of meter scale. Roots and shoots of plants of each treatment were weighed when fresh, and then kept in bamboo paper envelopes. The envelopes were left in an oven for 48h at 80°C and then weighed to obtain their dry weights. From each set, before drying galls were taken out and fixed in F.A.A. for histopathological studies.
NUMBER AND SIZE OF GALLS: The number of galls was counted by visual observation. The size of medium sized galls was obtained by measuring maximum length and width (in mm^) on a meter scale.
NUMBER OF MATURE FEMALES: Root samples taken from each treatment were blended with 200ml water in a warring blender for 30 seconds at low speed. The resultant suspension was passed through coarse and 100 mesh sieves in order to 116 separate root tissue. The total female population was counted with the help of counting dish. Total number of female nematodes in the suspension was divided by the weight of each root system to derive population per gram root.
SIZE OF MATURE FEMALE AND GIANT CELL: The mature females obtained from each treatment were placed on slide. Their images were traced on tracing paper with the help of camera lucida. The tracings were cut out and weighed. Tracing of 100^m^ on rice paper were also cut out and weighed. Weight of nematode tracings (Wl) were transposed to the weight of lOOfim^ tracing. In this way the area of the nematode Wl/W2xlOVni^ was calculated (Bird, 1970). In the same way, area of giant cell was obtained.
NUMBER OF EGG MASSES: Number of egg masses in infected roots were counted by staining egg masses with phloxin B. An aqueous solution of phloxin B 0.15g per lit' iBof water was prepared. Galled roots were placed in this solution for 15-20 minutes. Roots were rinsed in tap water. Red stained egg masses became observable and were counted easily.
NUMBER OF EGGS PER EGG MASS: About 10 mature egg masses were selected at random from root galls of each treatment. The egg masses were treated with 20ml of NaOCl (2%) solution and stirred vigorously for one minute. The eggs released from gelatinous matrix of egg masses were stained with acid fuchsin (Byrd, et. 117 al; 1972) and then counted under stereoscopic microscope.
REPRODUCTION FACTOR (RF) AND RATE OF
POPULATION INCREASE (RPI):
The rate of reproduction (Rf) and rate of population increase (RPI) gradually decreased with increase in initial inoculum level. Maximum being at the lowest and minimum at highest inoculum level.
HISTOPATHOLOGICAL STUDIES: Some galls, selected from each treatment were fixed in FAA and dehydrated through teritary butyl alcohol (T.B.A.) schedule (Johansen, 1940). The galls were infiltrated with paraffin wax after dehydration, and then embedded in paraffin wax. The embedded galls were trimmed to small blocks and then fixed on wooden blocks. About 10- 12nm thick sections were obtained with the help of rotary microtome. The sections were mounted on slides and kept in an incubator at 40°C for few hours (Johansen, 1940).The sections were stained with safranin and fast green as described by Sass, (1951). Anatomical details were observed under light microscope.
OBSERVATIONS ROOT AND SHOOT LENGTHS: Initial inoculum levels (P=5 J^, 50 J^. 500 J^ and 5,000 J^) of Meloidogyne incognita exhibited different suppressive effects on lengths of roots and shoots, and fresh and dry weights of roots and shoots of the 118 plant (Momordica charantia). Their impacts were more pronounced on roots than on shoots when compared with uninoculated (control) plants. In comparison to control, root and shoot lengths remained unaffected at P,=5 i^. With an increase in inoculum density to 50 juveniles per plant, an increase in length of both roots and shoot was observed. The increase, however, was non significant. At higher inoculum level (P^ = 500 J^), root and shoot lengths decreased (P<0.5) significantly. At the highest inoculum level, the lengths were drastically reduced (significant at P < 0.01) when compared with the length of control plants and with the lengths of the plants at rest of the inoculum levels. The lengths at P^=500 J^were significantly (P < 0.05) less, than at P^ ^ 5]^^ and P^ = 50 J^ (Table - 5). Reductions were higher in root lengths than in shoot lengths at P, = 500 J^ and at P, = 5,000 J^ (Table 5).
ROOT AND SHOOT WEIGHTS: Fresh weights of roots as well as shoots remained unaffected at lowest (P,=5 Jj) inoculum level. Both root and shoot fresh weights increased, though non significantly, at the initial inoculum level of P^=50 J^ per pot. Maximum reduction in fresh weight was observed at the highest inoculum level (P^=5,000 J^). The reduction in root and shoot weights at highest inoculum level was significantly higher (P < 0.01) in comparison to the weights of control plants and the plants at other initial inoculum levels. The dry weights of roots and shoots neither increased nor decreased at P_= 5 J^. Similarly, in dry weights, the trends of increase or decrease were similar to fresh weights. Root and shoot dry weights non significantly increased at P 119
= 50 J2 and significantly decreased at P, = 500 J^ (P < 0.05) and at P, = 5,000 J^ (P < 0.05), respectively.
While comparing nematode inoculated plants with each other, it was found that at higher (P_ = 500 J^) inoculum level root and shoot weights were significantly lower than the weights at P^ = 05 i^ and P^ = 50 J^ per pot, and higher than at P_ 5,000 J,. At highest inoculum level (P^ = 5,000 J^), root and shoot weights were significantly lower as compared wih the weights at P, = 5 J^ and 50 J^ (P < 0.01) and at P, = 500 J^ (P < 0.05) (Table 5).
NUMBER OF GALLS: The galls were scanty and almost unnoticeable at P^ = 5 i^ per pot. At the next higher inoculum level (P,=50 J^) few and prominent galls could be seen with the naked eye. At higher inoculum level (P^ = 500 J^), the gall number was significantly (P < 0.01) higher than that at P^ = 50 J^. The galling was much severe at the highest inoculum level (P^ - 5,000 J^) and was significantly higher than at other inoculum levels,(Table 5).
SIZE OF GALL: The size of gall significantly (P = 0.01) increased at higher inoculum levels (P, = 500 J^ and Pi = 5.000 J^) as compared to the galls at lower inoculum levels (P_ = 5 J^ and P^ = 50 J^). There was also a significant (P_ = 0.05) increase in gall size at P_ =^ 50 J2 when compared with that at P^ = 05 J . The galls attained maximum size at the highest P_ =5,000 J^ which were significantly (P = 0.01) larger than the galls at all the lower inoculum levels (Table 5). 120
NUMBER OF MATURE FEMALES: The number of mature females per gram root increased with an increase in initial population level. In comparison to the plants at P_ = 05 i^, the number of mature females recovered from plants at other inoculum levels was significantly higher. More females were collected from plants at P^ = 50 J^ than at P, = 5 J^. The number of mature females per gram root increased at P^ = 500 Jj and at P^ = 5,000 J2. Their values were maximum at the highest inoculum level followed by P, = 500 J^, P, = 50 J^ and P, = 5 J^ (Table 5).
SIZE OF MATURE FEMALES: A significant decrease (P < 0.05) in size of mature female was observed at P^ = 5,000 J^ when compared with the size at P_ = 5 J^. However, the average size was not affected very much with increase in initial inoculum levels. The difference in size of mature females at lower initial inoculum levels was non significantly different when compared with each other (Table 5).
NUMBER OF EGG MASSES: Number of egg masses per plant increased as the number of juveniles introduced per plant increased. At lowest inoculum level (P^= 5 J^) the number of egg masses recovered was as low as (2.52). More egg masses (43.8) occurred on roots having higher inoculum level (Pi = 50 J2). Egg mass number further increased at P^ = 500 J^, which was significantly higher than at P_ =5 J^ and P^ 50 ]^. Egg mass formation was maximum (243.30) at highest inoculum level. At this level, the number was significantly (P < 121
0.01) higher than that at any other inoculum level (Table 5).
NUMBER OF EGGS PER EGG MASS: Number of eggs per egg mass decreased with an increase in initial population level. However, the differences were not significant upto P^ = 500 Jj. A significant (P < 0.05) decrease was observed at P^ = 5,000 J2 when compared with all the lower inoculum levels (Table 5).
HISTOPATHOLOGY:
Root-knot nematode infection causes the formation of a group of multinucleate cells called giant cells. Second - stage juveniles of Meloidogyne incognita, after penetrating into the roots of Momordica charantia led to the induction of 6-8 multinucleate giant cells. The giant cells were induced in phloem region. A cluster of more than 10 giant cells was observed when two nematodes lying side by side were feeding at the same site. The gaint cells in the clusters varied in size from smallest to largest. (Fig. 32). The number of giant cells varied depending upon initial inoculum level, usually one female was found associated with one giant cell cluster, but occasionally more than two females were also found feeding at same site, at higher inoculum levels. Also, at lower inoculum levels, only one or two females were observed in parenchyma ray region when seen in transverse section (Fig. 33).
Density of the giant cell cytoplasm decreased with increase in initial inoculum level (Fig. 34). The giant cell cytoplasm was dense, more granular 122 and stained red at lower inoculum level (Pj = 5 J^ and 50 J^) (Fig. 35). Whereas at higher inoculum levels (Pj = 500 and 5,000 Jj) it was less dense and stained brown (Fig. 36 ).
At highest inoculum level (?^= 5,000 J^) significant (P < 0.01) reduction in the size of the giant cell was observed, when compared with giant cell at lowest (Pj = 5 Jj) initial inoculum level. However, there was gradual decrease in the size of gaint cell with increase in initial inoculum level (Table 5).
The large giant cells at higher inoculum level enclosed small amount of cytoplasm but the small and medium sized giant cells were almost empty (Fig. 37). At lower inoculum levels only the small sized giant cells (Fig. 38) were empty, medium sized contained little and large sized contained much cytoplasm. (Fig. 39).
The multinucleate condition of giant cells varied at different inoculum levels. The number of nuclei was higher at lower initial inoculum level (Fig. 35) but the number decreased and was lower at higher inoculum levels (Fig. 36). Size of the nuclei was independent of the inoculum level. The nuclei were amoeboid, circular, and oval in shape at lower inoculum but at higher inoculum level the nuclei were mostly of amoeboid shape. The shape and size of the nucleoli were not affected by the increase or decrease in inoculum density. The nucleoli varied from 4-10 in single nucleus, but 4-6 nucleoh were observed frequently (Fig. 40). Some females were observed 123 associated with large egg sacs at higher inoculum levels (Fig. 41), but the egg sacs were not prominent at lower initial inoculum levels (Fig. 42).
The quantity of abnormal xylem witnessed more at higher inoculum levels (Fig. 43), than at lower initial inoculum levels (Fig. 44). However, the xylem elements occupied larger area at higher inoculum levels than at lower inoculum levels as revealed by transverse section (Fig. 34). The distortion of xylem strands was more at higher inoculum level (Fig. 45) than at lower inoculum level (Fig. 46). Similarly the phloem strands were also less distorted at lower than at higher inoculum levels (Fig. 46 ). The giant cell cluster was found near the normal phloem strand and abnormal phloem. Their amount increased at higher than at lower inoculum levels.
DISCUSSION Plant response towards to Meloidogyne incognita could be reflected by the symptoms developed on the roots and the shoots. Alterations in root anatomy are peculiar characteristics to this specific type of host - parasite relationship. The most striking above ground symptom of root-knot disease is the stunting of the affected plants. However, it is not necessary that the plant would always develop the characteristic symptoms. It is evident from our studies that if primary inoculum comprised of a very low amount, it did not produce above ground symptoms. Although, juveniles entered the roots and caused gall formation but their overall effect on the plant growth was negligible. From our findings it might be concluded that low population 124 density of second-stage juveniles induced infection without causing any significant damage to the plant. Earlier workers like Tyler (1933), Dropkin (1954), and Dropkin and Boone (1966) used single larval inoculation to find its response at the site of infection. They did not mention its effect on the growth of the plant.
Wallace (1971) hypothesized that lower population densities of Meloidogyne spp. stimulated the plant growth. Our studies carried out on Momordica charantia confirm his findings. In presence of second-stage juveniles, an initial inoculum level of 50 J^ per pot enhanced plant growth. Root and shoot lengths, and their weights increased at this level. This response might be due to the presence of certain growth hormones. Auxin and cytokinin (Setty and Wheeler, 1968; Balasubramanian and Ranga swamy, 1962) have been ascertained near the nematode head that probably stimulated the plant growth.
At both higher inoculum levels (P^ = 500 and P^ = 5,000 J^) growth of the plant suppressed significantly. The damage to the plant, by Meloidogyne spp.. involves several mechanisms. The plant growth might be affected due to removal of nutrients by the nematode. The nutrient transport from the root towards shoot is hampered due to anatomical abnormalities in the galled regions. Moreover, photosynthates are diverted towards the giant cells that act as metabolic sinks for the organic compounds. All these malfunctions contribute in suppressing plant growth and yield (Hussey, 1985). Root-knot 125 nematode infection markedly retards the rate of absorption by the roots and also affects the rate of translocation towards the shoot apices. Oteifa (1952) compared reduced shoot growth of Meloidogyne infected plants with the plants growing in nutrient deficient soils. He observed nutritional deficiency symptoms in the foliage of plants infected with root-knot nematode. Hunter (1958) correlated poor nutrient uptake and suppressed plant growth with highly reduced root system as a result of root-knot nematode infection.
Increase in amount of initial inoculum level of M mcog«/7o was responsible for increased loss in fresh and dry weights of Momordica charantica. Bird (1962, 1968) opined that the rate of photosynthesis in tomato decreased with the increase in initial inoculum level. However, Olthof and Potter (1972) reported an inverse correlation between Meloidogyne hapla density and crop yield of certain vegetables. They estimated commercial losses upto 46% and 64% for onion and potatoes, respectively, at an inoculum level of 28,000 J^ per plant. Similarly, Loveys and Bird, (1973) reported decline in net photosynthetic rate within two days after inoculation at high inoculum levels of M javanica on tomato. Barker (1977) assessed yield losses from 3.7 to 19.9% due to M arenaria, M. hapla, M. incognita and M javanica for each ten fold increase in initial densit>' for each species. Samathanam and Sethi, (1996) observed maximum reduction in shoot length, fresh and dry shoot weight at 8,000 J^ with an increase in inoculum level from 500-8000 J^. The reduction in growth characteristics was not proportionate to the increase in inoculum level. 126
The present pathogenicity test with M incognita on bitter gourd revealed that plant growth characters like root and shoot length, their fresh and dry weights were found to be adversely affected as the level of inoculum increased from 500 to 5,000 J^. The reduction was significant (P < 0.01) in length and weight of the plants at the highest inoculum level (P^ = 5,000 J^), as compared to uninoculated plants. Adverse effects on growth of different plants with an increase in primary inoculum level oiMeloidogyne spp. have been reported by several workers (Christie, 1936; Krusberg and Nielsen, 1958; Wallace, 1969; Ferris, 1974; Barker and Olthof, 1976; Nordacci and Barker, 1979; Kinloch, 1980, 1982; Rodriguez-Kabana and Williams, 1981; Appel and Lewis, 1984; Ibrahim and Lewis, 1985; Fazal et. al. 1996). Seinhorst (1960) observed a measurable damage occurring only when the nematode population density exceeds a certain limit. In the present study the growth parameters and nematode inoculum density showed positive linear relationship. Wallace (1963) and Oostenbrink (1966) opined that the increase in nematode populations and subsequent reductions in the yield of crops or other manifestations of pathogenic effects are directly influenced by initial density of the nematode in soil.
With an increase in primary inoculum level not only gall number but also size of the gall increased as is evident from data (Table 5). This trend might be due to the fact that at higher inoculum levels, large number of juveniles explored new feeding sites which resulted in increased number of galls on the infected roots. At higher inoculum level, enormously large galls 127 were formed, because many juveniles penetrated the same feeding site and caused multiple hypertrophic and hyperplastic reactions. Ibrahim and Lewis (1985) also observed enhanced gall number in soybean roots with corresponding increase in initial population levels of M incognita. Similarly, Fazal et. al. (1996) reported gradual increase in number of galls per plant (158-311) with the increase in initial inoculum level of M. inocgnita in black gram. Size of the gall increases with an increase in inoculum level (McClure and Viglierchio, 1966; Arens et. al. 1981).
Dropkin, and Boone (1966) observed a large portion of the gall associated with egg masses. As is revealed by our data the egg masses per plant increased with the increase in inoculum level. It is quite reasonable that lower the inoculum level, lower will be number of mature females in the gall and consequently fewer the egg masses; and higher the inoculum level correspondingly higher will be the number of mature females as well as egg masses. Number of eggs per egg mass significantly decreased at the highest inoculum level, however, at lower inoculum levels the differences in the number of eggs per egg mass were non significant. It seems that size of the mature female and the number of eggs produced by it are interrelated. The frequency of eggs per egg mass per mature female might be considered as a measure how the development of female and egg mass production is influenced by the nematode density. Limited food and space, probably, caused detrimental effects on the maximum development of the nematode and consequently on egg mass production. Insufficient nutrition seems to 128 be the main reason behind the low production of egg masses.
Momordica charantia roots in response of feeding by M. incognita juveniles undergo pronounced anatomical changes. Paramount among these changes is the development of elaborate permanent feeding sites, called giant cells. Our studies reveal thatM incognita induced 6-8 multinucleate giant cells in vascular tissues of M charantia. The number of giant cells increased to more than 12 when two nematodes lying side by side started feeding at the same feeding site. Christie (1936) reported 3-6 giant cells in tomato, similarly 4-9 giant cells were reported in sweet potato, (Krusberg and Nielsen, 1958) 2-5, 10-12 giant cells in Gardenia, (Davis and Jenkins, 1960) 5-9 in soybean (Dropkin and Nelson, 1960); 4-7 in Hibiscus (Littrell, 1966), 4-5 in tifdwarf, (Heald, 1969); 3-5 in barley (Ediz and Dickerson, 1976); 5-6 in Impatiens (Jones and Payne 1978). The shape of giant cells, as observed in transverse and longitudinal sections was circular, ovate to oblong. The giant cells were generally oblong because, according to Christie (1936) they arise from elongated cells which in normal development lead to the formation of vessels. The giant cells adjacent to the nematode head are larger as compared to those which are away from the nematode. Contrary to this finding our conclusion is that, the gaint cells are formed from the cells that under normal conditions would develop into phloem elements as has been proposed by Byrne et. al. (1979) and Hisamuddin and Siddiqui (1992). 129
The giant cells are produced as a result of continuous stimulus received from the nematode (Bird, 1962). The giant cell nearest to the nematode head gets direct stimulus and hence is larger than that which is away, and receives an indirect stimulus. Thus, formation of giant cells is essential for a successful host parasite relationship. Tissue preferred for the formation of giant cells is primary phloem or adjacent parenchyma (Christie, 1936; Krusberg and Nielsen, 1958; Byrne, et. al., 1979). The figures reveal that the site where the giant cells are produced is occupied by parenchyma ray during normal development of the root. The protophloem, is crushed and pushed towards the periphery as a result of secondary growth. Formation of giant cells and development of the nematode, in the protophloem, exert considerable pressure on the surrounding tissues. This results in abnormalities in the neighbouring cells and tissues. At higher inoculum levels average size of the giant cell reduces. It might be because at high inoculum density all the parenchyma rays are occupied by the giant cells and the nematode. More than one nematodes cause multiple induction of giant cells in a limited space.
Bird and Loveys (1975), McClure, (1977); unequivocally asserted that Meloidogyne acts as a metabolic sink in diseased plants. The increased metabolic activity of giant cells stimulates mobilisation of photosynthates from shoots to roots and particularly to the giant cells where they are removed and utilised by the feeding nematode. In our experiment, at the 130 lower inoculum level, dense cytoplasm in giant cells indicated that metabolites were sufficiently supplied to the giant cells by the plant. At higher inoculum levels, emptiness and much vacuolation in giant cells showed that either supply of photosynthates was not enough, or there was more demand by the nematodes than its rate of formation. Higher number of nuclei in the giant cells, at lower inoculum level, expressed greater metabolic activity of giant cells. At higher inoculum level emptiness of giant cells does not mean lower metabolic activity but speedy removal of metabolities by the nematodes. The higher number of deeply stained nuclei, in giant cell, evidenced the higher metabolic rate at lower inoculum level. On the other hand light stained nuclei at higher inoculum level indicated a low metabolic rate of the giant cell due to scarcity of food.
Abnormalities in xylem as a'result of root-knot infection has been reported in almost all the histopathological studies. At higher inoculum level several nematodes induce giant cells at a particular site, resulting in multiple hypertrophic and hyperplastic reactions, and also formation of abnormal xylem at various sites. Probably, combined effects of these reactions stimulated abnormal xylogenesis. Increase in number of phloem elements was probably due to the formation of higher number of giant cells that was essential for regular supply of food to the giant cells. Hyperplasia not only increases parenchyma but also phloem elements.
Thus, with an increase in initial inoculum level, length and weight of 131 plant decreased, number and size of galls, number of mature females per gram root, and number of egg masses per plant increased and the size of giant cell and number of eggs per egg mass decreased. Histological studies revealed that giant cell cytoplasm was dense at lower than at higher inoculum level. There was higher number of nuclei in the giant cells at lower inoculum level. At higher inoculum level amount of abnormal xylem and phloem elements increased. EXPERIMENT 4.
Histopathological responses of selected varieties of Momordica charantia to Meloidogyne incognita.
Different varieties of bitter gourd {Momordica charantia) are grown in Aligarh district alongwith other cucurbitaceous vegetables. Most of the varieties are highly susceptible to Meloigogyne species that cause heavy losses to it and other vegetables. A few species, however, are less susceptible. As far as literature about work on bitter gourd, in relation to phytoparasitic nematodes is concerned, few mentionable reference have been traced. Pankaj and Siyanand (1992) tried to control root-knot disease on bitter gourd through chemical methods. Histopathological studies are yet to be done.
Following experiment was aimed at determining the responses of different varieties of M. charantia towards Meloidogyne incognita. Six varieties were collected from various seed sources to evaluate the degree of susceptibility or resistance, if any, against the nematode.
MATERIALS AND METHODS The seeds of bitter gourd {Momordica charantia) vars. (1). P.D.M. National Seeds Corporation, New Delhi. (2). Faizabadi (long green) Indian Seeds Corporation, Delhi. (3). Jhalarwali (New Rama Seeds Corporation, N. Delhi and (4). Jaunpuri, - Alpha Seeds Pvt. Ltd., Delhi. (5). Baramasi, 133
Aligarh Seed Company, Aligarh (6) Aligarh local, Aligarh Seed Company, Aligarh, were axenised by NaOCl method (Koenning and Barker, 1985). The seeds of each variety were allowed to germinate on moist Whatman filter paper in sterilized petri dishes of 10 cm diameter. The petri dishes were kept in an incubator at 30°C. After three days when the seeds sprouted, they were transferred to 30 cm diameter clay pots filled with autoclaved soil in the ratio of 7clay : 3 sand : 1 manure.
After one week the seedlings of each variety were inoculated with 10, 100, 1000 and 10,000 freshly hatched second - stage juveniles of Meloidogyne incognita as described in detail in Experiment no 1. There were five replicates for each treatment. Each set also comprised of uninoculated plants which served as control. The pots were kept in glass house at 30-35°C and were arranged in randomized complete block design. The plants were harvested 45 days after inoculation. The data for different parameters viz. length, fresh and dry weights of roots and shoots, number of egg masses per plant, number of eggs per egg mass, number of galls per plant, size of gall, number of mature females per gram root, size of mature females and size of giant cell were calculated as described in Experiment no.l. For determining gall index 0-5 scale was used (Sasser, er. al., 1984).
For final population (Pf), the soil population was estimated by Cobb's decanting and sieving method and root population was estimated by Blender- Baermanan tray method (Hooper, 1985). 134
Reproduction factor (Rf) was calculated by the formula: Pf Rf = Pi
Where Pf is the final ppulation and Pi is the initial population. Rate of population increase (RPI) was calculated by the formula. Pf-Pi RPI = Pi
Taylor and Sasser's rating scale for the presence of root-knot nematode galls or egg masses on roots.
Number of galls or egg masses Gall index (GI) or Egg mass index (EI)
0 0 1-2 1 3-10 2 11-30 3 31-100 4 100 + 5
For histopathological studies galled roots were collected from each variety maintained at an inoclulum level of 1,000 juveniles per pot. The galls were washed free of soil, fixed in F.A.A. (formalin aceto alcohol), dehydrated through T.B.A. schedule, infiltrated and embeded in wax as described in detail in Experiment No. 1. Sections of 10-12 ^im thickness were obatined with the help of rotary microtome and stained with safranin and fast green (Sass 1951). Anatomical details were observed under light microscope. Photographs of the relevant sections were taken. 135
OBSERVATIONS ROOT AND SHOOT LENGTH: From the data it is evident that uninoculated Faizabadi variety of Momordica charantia exhibited luxuriant growth with long roots and long shoots. The plants of variety Aligarh local exhibited poor growth in comparison to other varieties. At different inoculum levels the responses of all the varieties were different against Meloidogyne incognita.
The root and the shoot lengths of Faizabadi remained unaffected by the nematode infection upto the initial inoculum levels of P_= 1,000 J^. At the highest inoculum level (P^ = 10,000 i^ both root and shoot lengths decreased, non significantly. The variety Jhalarwali, exhibited reduction in root and shoot lengths at P^= 1,000 J^ per plant; the reduction was significant (P < 0.05) at highest (P_ = 1,0000 J^) level only. In PDM and Jaunpuri no significant change was observed at Pj = 10 and P_ = 100 J^ per plant. At P^ = 1,000 Jj and P, = 10,000 J^, significant reductions (P < 0.05 and P < 0.01, respectively) were detected. The varieties Baramasi and Aligarh local remained healthy at P = 10 Jjper plant. Both varieties exhibited non significant increase in lengths of both root and shoot at P = 100 J^ per plant. At 1.000 J^ and 10,000 J^ per plant, the root and the shoot lengths of these varieties reduced. .AJigarh local was the most affected variety (Table 6, 7. 8. 9, 10 «fe 11).
FRESH WEIGHT OF ROOTS AND SHOOTS: Fresh weights of roots and shoots of the variety Faizabadi, when 136 compared with control, remained more or less unchanged at lower (P, = 10 and 100 J^) and higher (P, = 1,000 J^) inoculum levels. The fresh weights decreased at highest (P; = 10,000 Jj) inoculum level but reduction was non significant. In the variety Jhalarwali, reduction was not observed at lower inoculum levels. The weights decreased at higher (P^ = 1,000 J^) and highest (P^ = 10,000 Jj) inoculum levels. Reduction in weight was significant (P = O.Oj) at the highest inoculum level only (Table 6, 7).
In presence of 10 juvenils per pot, deleterious effects of root-knot nematode were not observed in the varieties PDM and Jaunpuri. At the inoculum level of P^ = 100 J^, root and shoot weights markedly increased, when compared with control. The increase, however, was non significant. At higher (P^ = 1,000 J^) inoculum level a significant (P < 0.05) reduction in weight was observed. Fresh weights of roots and shoots further decreased at highest inoculum level (P^ = 10,000) which was significant at one per cent level, when compared with control (Table 8, 9).
The varieties Baramasi and Aligarh local were not affected by the nematode at lowest (P^ = 10 J^) inoculum level. The root and shoot weights at P^ = 100 J^ were non - significantly higher when compared with the control. The fresh weights significantly (P <0.01) decreased at P_= 1000 Jj. Reduction in weight was heavy and also significant (P < 0.01) at highest inoculum level (P, = 10,000 J^) (Table 10, 11). 137
DRY WEIGHTS OF ROOTS AND SHOOTS: Corresponding to fresh weights of roots and shoots, their dry weights also exhibited similar trends. In comparison to control, the dry weights of roots and shoots of the variety Faizabadi, at lower (P^= 10 }^ and 100 J^) inoculum levels and at higher (P. = 1,000 J^) inoculum level, were the same. The dr>' weights of both roots and shoots decreased at highest inoculum level (P^ = 10,000 J^). The reduction, in comparison to control, was non significant. The variety Jhalarwali exhibited significant reduction (P < 0.05) at 1,000 Jj and (P < 0.01) at 1,0000 J^ level (Table 6, 7).
Dry weights, of Pusa Do Mosami and Jaunpuri, neither decreased nor increased at lowest (P^ = 10 J^) inoculum level, in comparison to control. The weights of both roots and shoots increased slightly but non significantly at the next incoulum level of P^ = 100 J^ per pot. Their weights decreased significantly (P < 0.5) at the higher (P^ = 1,000 J^) inoculum level. The weights of roots and shoots sharply and significantly (P < 0.01) decreased at the highest inoculum levels (Table 8, 9).
Baramasi and Aligarh local varieties remained unaffected at the lowest inoculum level, when compared with control. An increase in dry weights of roots and shoots was observed at the inoculum level of 100 J^ per pot. This increase was non significant in comparison to control. At higher inoculum level (P_ = 1,000 J^), and (P = 10,000 J^) dry weights of both roots and shoots considerably and significantly (P < 0.01) decreased (Table 10, 11). 138
GALL NUMBER AND SIZE: In variety Faizabadi, no galls were observed at lower initial inoculum levels (P^ = 10 and 100 J^). Occasionally small sized galls were observed at (P^ = 1,000 Jj) higher inoculum level. At highest inoculum level, the galls were few (18.74) and were smaller in size (3.67 mm^). The variety Jhalarwali had no galls at P, = 10 J^ and P, = 100 J^. The galls were observed at 1,000 J^ inoculum level which were larger than occurring on Faizabadi. At highest inoculum level, the gall number increased as compared to Faizabadi at the same inoculum level. The galls were not very large in size (Tables 6, 7).
In all other varieties the galls were observed at all the initial inoculum levels. The varieties PDM and Jaunpuri produced 57 and 65 galls at P^ = 100 Jj. The gall number increased to 107.00 and 126.11, at higher inoculum (P^ = 1,000 Jj) level. At highest inoculum level gall numberwas as high as 159 and 170.05 galls per plant, respectively. At higher and highest inoculum levels, the galls were larger in size (Tables 8, 9).
The galls were few and small at P = 100 J^, in Baramasi and Aligarh local varieties. The gall number was high at P^ = 1000 J^, 171 galls per plant in Baramasi and 227 galls per plant in Aligarh local. Their size was large in both the varieties. At the highest inoculum level the gall number per plant was 217 in Baramai and 241 per plant in Aligarh local. In the variety Aligarh local, the number, as well as the size of the gall was maximum as compared to other varieties (Table 10, 11). 139
NUMBER AND SIZE OF MATURE FEMALES: In the varieties Faizabadi and Jhalarwali, galled tissues were not observed at P. = 10 J^ and 100 J2. At higher (P.= 1,000 i^ per plant) inoculum level, the galls were rarely present and their size was very small. In these galled tissues, mature females were in small number, 3.46 per gram root. In most of the galled portions, the females were found in different stages of development. At highest (P_ = 10,000 J^) inoculum level, no mature females were detected. The nematodes were in various developmental stages.
Nematodes were not observed in Jhalarwali at lower (P = 10 J^ and 100 J^) inoculum levels. In the galled regions of roots at P^ = 1,000 J^, 4.2 mature females per gall were observed. Their size was 6.57^m'*. At highest (P_ = 10,000 Jj) inoculum level the number of galls as well as number of females, (15.1 per gall). At both initial inoculum level P^ = 1,000 J^ and 10.000 Jj, most of the nematodes were in developing stages. In the varieties PDM and Jaunpuri 19.75 and 22.88 mature females per gram root were found respectively , at P^ = 100 J^ per plant. Their number increased to 27.01 and 30.21 at 1,000 J^ per plant, initial inoculum level. At highest inoculum level (P^ = 10,000 J,), their number was much higher comprising of 56.10 and 61.21 mature females per plant. The size of mature female at all the inoculum levels was the same and was approximately about 8.11 and 8.5 l^im".
Number of females in Baramasi and Aligarh local was higher in cmparision to other previously mentioned varieties, at respective initial 140 inoculum levels. The numbers of mature females recovered per gram rootwas 30.17, 57.23 and 75.85 at P. = 100 , 1,000 J^ and 10,000 Jj, respectively, in Baramasi. In Aligarh local, theirnumber was 30.01, 55.71, and 78.85 atPj = 100 J„ 1000 Jj and 10,000 J^, respectively. The size of mature female was around 9.95 and 9.%l\im^ at 100 J^ and 9.65nm2, 9.32^im2 at 1,000 J^ and 9.55 and 9.15fim^ at 10,000 J^in Baramasi and Aligarh local respectively.
GALL ANATOMY: In Faizabadi variety, the root anatomy was normal at P^ = 10 J^, and 100 Jj per plant. At 1,000 J^, inoculum level certain portions of root exhibited galling effect. Hypertrophied and hyperplastied cells occurred in these regions. The giant cells were small, containing less, and highly vacuolated cytoplasm. The nuclei in the giant cells, were few. Hyperplastic parenchyma occupied smaller area. No disruption in vascular tissues was observed (Fig. 47). Near the giant cells, abnormal xylem elements were few. The nematodes associated with the giant cells were not mature, but were in developing stages (Fig. 48). At highest (P. = 10,000 J^) inoculum level, the galls were prominent. The galled roots, in transverse and longitudinal sections, revealed small sized giant cells, less amount of hjperplastic parenchyma and abnormal xylem, and less disruption in vascular tissues (Fig. 49). In some roots (Fig. 50) necrotic tissues were observed, but neither the nematode nor the giant cells could be traced in the affected zone. 141
The roots of Jhalarwali were also free of galls at P^ = 10 J^ and 100 Jj. Galling was conspicuous at Pj= 1,000 J^ and 10,000 i^ per plant. In the galled regions, the giant cells were smaller and the nematodes were in various stages of development (Fig. 51). Hyperplastic parenchyma and abnormal xylem were more as compared to Faizabadi variety. At higher inoculum level (P^ = 1,000 J^), there was less disruption in vascular tissues (Fig. 52). At the higher inoculum level (P. = 10,000 J^), developing females with small sized giant cells were frequently observed (Fig. 53). Some females, associated with egg masses were also occasionally seen (Fig. 54).
In all the other varieties more disruption was seen in vascular tissues, in galled roots. The number of females and the number of giant cell complexes were higher. Hyperplastic parenchyma and abnormal xylem also occupied larger area. At lower inoculum level (P. = 100 J^), in PDM variety, few nematodes with prominent giant cells were observed when seen in transverse sections (Fig. 55). The females were all mature and were associated with egg masses. The giant cells were large, multinucleate and filled with dense and granular cytoplasm (Fig. 56). Abnormal xylem near the giant cells was more. At higher inoculum level (P. = 1,000 J^) more females were seen causing the formation of giant cells, abnormal xylem and abnormal phloem. At highest inoculum level (P^ = 10,000 J^) higher disruption in the root anatomy was observed (Fig. 57).
In the variety Jaunpuri, few nematodes were found at low inoculum level (P^ = 100 Jj). As seen in trasnvere sections (Fig. 58), the deformities 142 in the root nanatomy were less. Quantity of normal xylem was more as compared to abnormal xylem. At higher inoculum levels (P^ =1,000 and 10,000 Jj), the number of females per gall increased causing heavy disruption in vascular tissues. The giant cells were large and were surrounded by abnormal xylem (Fig. 59).
The variety Baramasi also exhibited few females, as seen in transverse section (Fig. 60). At higher inoculum level (P^ = 1,000 J^) number of nematodes, giant cells, amount of abnormal xylem and phloem increased (Fig. 61). At the highest inoculum level a large number of females were found. Both mature and developing females were seen causing heavy damage to normal tissues of their root (Fig. 62). In Aligarh local at lower inoculum level (P^ =100 Jj), fully mature nematodes (alongwith their egg masses) associated with giant cells were observed (Fig. 63). At this level the giant cells were quite prominent and were filled with dense cytoplasm (Fig. 64). At higher Pi, the number of females was more and therefore there were more abnormal tissues and less normal tissues (Fig. 65). At the highest P^, females occupied almost all parts of the tissues as is evident from the (Fig. 66).
DISCUSSION
Momordica charaniia unlike other cucurbits has some medicinal properties and contains alkaloids like momordicine and has some bitter principles which are different from other cucurbits (Sastri, 1962). Keeping 143 in mind this characteristic, six varieties of M charantia were collected from various sources and were tested for their effect on the Meloidogyne incognita and vice versa. The responses of M incognita were not uniform as is evident from the data (Table 7, 8, 9, 10 & 11) and the anatomical studies.
Longer roots and shoots of the variety Faizabadi at all the initial inoculum levels, as compared to other varieties exhibited rapid growth. From this observation it might be inferred that either rate of elongation and maturation of the roots is so fast that the juveniles do not reach easily to the growing zone, or the roots emanate certain nematode repelling substances, that keep the nematodes away from the roots. Crittenden (1954) suggested that woody nature of roots might make the plant resistant against root-knot nematode. The variety Jhalarwali showed relatively higher reduction in root and shoot length. At the highest (P. = 10,000 J2) inoculum level higher reduction indicated susceptibility towards the nematode.
The varieties PDM and Jaunpuri did not show any change at P^ = 10 Jj and Pi 100 J^. The root and shoot lengths decreased significantly at 1,000 Jj and 10,000 J2. This shows that these two varieties are more susceptible than Faizabadi and Jhalarwali. The varieties Baramasi and Aligarh local were found to be the most susceptible as reductions in their lengths were much higher as compared to other four varieties, at P. = 1,000 and P = 10,000 Jj. Both these varieties exhibited slight but non-significant increase in plant length at P_ = 100 J^. Probably at this level, the growth hormones produced at the time of begining of infection contributed in enhancing the growth. 144
From the data of fresh and dry weights of roots and shoots it is evident that the variety Faizabadi was not affected significantly by the nematode. It seems that Faizabadi variety is resistant against the nematode. The variety Jhalarwali showed non-significant reduction in weights at R = 1,000 J^and significant (P < 0.05) reduction at P^ = 10,000 J^. This indicates that the variety Jhalarwali is neither resistant nor very susceptible but it resists against root- knot nematode infection at certain initial inoculum level. Probably this variety is tolerant against M incognita.
At 100 Jj inoculum level, it was observed that four varieties viz., PDM, Jaunpuri, Baramasi and Aligarh local exhibited an increase in dry and fresh weights of both roots and shoots. An increase in length, fresh weight and dry weights of roots and shoots might be due to certain chemicals produced after infection which stimulated plant growth as was also reported by Veech (1981). Low inoculum density has often been reported beneficial for the plant growth. Wallace (1971) reported enhanced root and shoot weights of vegetable plants of cabbage, tomato and potato. Madamba et. al., (1965) also reported stimulated growth of non-host plants. The growth of another cucurbit Luffa aegyptica var. Ghiya was also found to be increased at low inoculum level of M incognita (Hisamuddin and Siddiqui, 1992). In our experiments the later four varieties showed increased plant growth at low inoculum level.
Fazal et. al, (1996) suggested a threshold level of 1000 J^ of M incognita on mung bean and at higher inoculum levels the effects were 145 detrimental to plant growth. It is evident from our studies that in the four varieties under study, PDM, Jaunpuri, Baramasi, and Aligarh local, the plant growth in terms of fresh and dry weights of roots and shoots significantly decreased at P. = 1,000 J^. At further higher inoculum level (P^ = 10,000 J^) all the growth parameters, i.e., root and shoot length, their fresh and dry weights signficantly decreased. In the presence of higher number of nematodes the chances of infection become higher. The growing roots as well as newly emerged roots are frequently attacked by the juveniles causing severe infection in roots leading to heavier damages to the plants. Contrary to this, Canto - Saenz (1984) reported better growth and yield of potato (Ej) in presence of higher nematode density. This was probably due to the incompatible response between the host and the nematode. In our experiments Aligarh local exhibited maximum reduction in plant grov^h and can be considered as highly susceptible variety.
Among six varities, Faizabadi and Jhalarwali did not develop galls on their roots, at lower inoculum levels (P; = 10 and 100 J^). This was probably because the larvae did not enter the roots or if entered they could not induce hypertophy and hyperplasia. At higher (P. = 1000 J^) inoculum levels occurrence of galls does not necessarily mean that the varieties are susceptible. At highest inoculum level the galls produced on Faizabadi were few in number and were very small. It might be due to invasion by a large number of juveniles at one feeding site. In the variety Jhalarwali, galls were more in number and larger than Faizabadi. It indicates that Jhalarwali is not 146 as resistant as Faizabadi.
In other four varieties, galls were observed at all the initial inoculum levels. The gall number as well as the gall size increased from the variety PDM to Aligarh local. This ascending trend indicated that the degree of suceptibility increased from PDM to Aligarh local. As our observations reveal, Aligarh local variety is highly susceptible towards M. incognita followed by Baramasi then Janupuri and lastly PDM. The variety PDM may be rated as moderately susceptible. According to Struble et. al. (1966), in resistant varieties there is less galling as compared to susceptible varieties.
Histopathological studies revealed that in Faizabadi variety fully developed nematodes were lacking. The nematodes traced so far were in various developmental stages. At certain locations deformed developing females were observed. Gentile et. al. (1962) also reported developing females in galls of resistant plants. Mc Clure et. al. (1974) found that either the nematode were in developing stages, or fragmented when their development stopped. Sosa-Moss et. al, (1983) did not find any nematode in the affected regions.
In the variety Jhalarwali, most of the females were in developing stages and only a few reached to maturity. It means that this variety was not as much resistant as Faizabadi. Other varieties probably provided favourable environment for nematode development because from rest of the four varieties mature females were recovered. 147
From the data, it is evident that in the variety Faizabadi, the nematode did not produce egg masses. It might be due to delayed development of the nematode.This also ascertains resistant nature of the variety. In the variety Jhalarwali formation of egg masses indicates favourable response of the host towards the nematode. Formation of egg masses in higher number in Baramasi and Aligarh local indicated susceptibility of the two varieties. In potato, Struble et. al (1966) reported few eggs per egg mass in resistant variety while McClure et. al. (1974) observed few egg masses on infected roots of resistant cotton cultivar. In our experiment, the variety Faziabadi did not allow the nematodes to develop into mature stage that inhibited egg mass production.
The roots of Faizabadi variety attacked by the nematodes showed necrotic regions. As reported by various authors necrosis in affected tissues is a common hypersensistive response of the plant (Gimalva, 1963; Malo, 1965; McClure et. al., 1974; Sawhney & Webster 1975; Sosa Moss et. al, 1983) Anatomical studies also revealed necrotic regions where the nematode attacked but could not develop to mature stages in the roots of Faizabadi. In some sections nematode fragments were also detected. The nematode, if enters in the roots of a resistant variety, it dies, disintegrates, and finally disappears (McClure er. al. 1974; Canto-Saenz, 1984).
The giant cells either did not develop or there were very small sized giant cellsin the infected roots of Faizabadi variety. Malo (1965) reported 148 empty giant cells after the death of Meloidogyne javanica infecting peach root stocks. In root-knot nematode resistant sweet potato, Struble et. al. (1966) found few or no giant cells where the nematode attacked the root. In Faizabadi variety, non induction of giant cells led to the death of the nematode. In the roots where giant cells were small, the development of the females was also slow. Probably small giant cells are not sufficient enough to fulfil the nutrient demand of the developing nematodes. Similarly, in Jhalarwali variety the giant cells were larger than those observed in Faizabadi but were not as large as in other four susceptible varieties.
In longitudinal sections of the roots of Faizabadi variety, it was observed that vascular tissues were not disrupted. However, in other varieties, orientation of vessel elements, and phloem elements changed because of hyperplastic and hypertrophic reactions in the affected parts. Several females were found associated with various giant cell complexes in the susceptible varieties. Amount of abnormal xylem, in the resistant variety Faizabadi, was small as compared to other susceptible varieties. SECTION-III
EXPERIMENT 5.
Origin of giant cells, development of galls and histology with special reference to xylem and phloem, of galled roots of Momordica charantia infected with Meloidogyne incognita
Root cells of different plants respond quickly and characteristically towards the juveniles oi Meloidogyne incognita (Littrell, 1966). The second - stage juveniles of Meloidogyne spp. penetrate and establish in roots (Christie, 1936). Some parenchyma cells become hypertrophied and multinucleate which are known as giant cells. Penetration in young roots takes place intercellularly towards the region of vascular differentiation that leads to giant cell induction and gall formation (Endo and Wergin 1973, Jones and Payne, 1978). Gall formation in tomato roots was observed even when the juveniles were outside the tomato roots (Schuster and Sullivan, 1960). Meloidogyne spp. infection accompanies cortical and stelar proliferation (Davis and Jenkins, 1960), and hypertrophy and hyperplasia in cortex, pericycle and stele of soybean roots (Ibrahim and Massoud, 1974).
Giant cells are generally formed from undifferentiated vessel elements or from xylem parenchyma (Christie, 1936, Hodges and Taylor, 1966) or from provascular strand (Krusberg and Nielsen, 1958 and Littrell, 150
1966). The giant cell formation has also been reported from protophloem cells. (Byrne, 1973). The present work was carried out (i) to investigate the origin and development of gaint cell and histology of galls in Momordica chrantia roots infected with Meloidogyne incognita, (ii) to study abnormalities in xylem and phloem, and (iii) to find out any relationship between the giant cells and the vascular elements.
MATERIALS AND METHODS
COLLECTION AND MAINTENANCE OF Meloidogyne incognita CULTURE:
A single egg mass obtained from Meloidogyne incognita infected egg plants was axenised by placing in 0.5%NaOCl solution for five minutes. It was washed thrice with sterilised distilled water and allowed to hatch at 27°C in an incubator. Egg-plant seedlings raised in 25cm pots containing autoclaved soil were inoculated with the juveniles thus obtained. In order to maintain sufficient inoculum throughout the course of investigation new egg-plants were inoculated with at least 15 egg masses obtained from pure culture.
AXENIZATION OF SEEDS:
The seeds oiMomordica charantia were axenised by NaOCl method (Koenning and Barker, 1985). About 200 seeds were poured into a sterile 151
beaker. The beaker was filled with a 1:1 mixture of 95% ethanol and 5.25% NaOCl. The mixture was stirred gently and allowed to soak for about 8-10 minutes. The mixture of alcohol and NaOCl was drained out and the seeds were rinsed with sterile distilled water.
SEED GERMINATION AND TRANSPLANTATION OF SEEDLINGS :
About 10-12 axenised seeds were placed on a moist, sterilised filter paper kept in a sterilised petri dish. The seeds were allowed to germinate for three days. The germinated seeds were transferred to 15cm diameter clay pots filled with clay, sand, and manure in the ratio of 7:3:1.
INOCULATION:
The egg masses, hand picked from the egg plant root galls, were allowed to hatch and the second-stage juveniles were collected in sterilized distilled water, and counted with the help of counting dish.
When the seedlings became one week old, holes of 5-7 cm depth around the plants within a radius of 2 cm from the plant were made and through these holes second-stage juveniles (1000 J^ per 10 ml DDW per pot) were introduced with the help of sterilised pipette. The holes were plugged with sterilised soil. To maintain soil moisture, the pots were regularly watered. Non inoculated plants served as control. 152
HARVESTING:
Five seedlings were harvested at an interval of 24 h to 72h. Then on 6th day and after a gap of 6 days until the 30th day. The roots were washed thoroughly but gently under tap water to remove all soil particles. The galled roots were cut into small pieces and processed.
PROCESSING:
(1). Fixation, (2). Dehydration, (3). Infiltration, (4) Embedding, (5). Sectioning (Ribbon Mounting), and (6). Staining, have been described in detail in experiment 1.
The pieces of galled roots and healthy roots were immersed in a fixative of formalin-aceto-alcohol F.A.A, for two to three days and dehydrated through T.B.A. The dehydrated galls were infiltrated with paraffin oil and wax and then embedded in paraffin wax. The galls along-with wax were shaped to small blocks by trimming. Sections of 10-12 |im were obtained with the help of rotary microtome. The sections were mounted on glass slides and stored (Johansen, 1940) for two to three days. The sections were stained with safranin and fast green (Sass, 1951). These sections were studied under light microscope and necessary photographs were taken. 153
OBSERVATIONS
A. GIANT CELL
24 h After Inoculation:
The second stage juveniles of Meloidogyne incognita penetrated the young root tips of M. charantia at or just behind the root cap. Soon after entering, the juveniles migrated towards the differentiating zone of root tip, pushed the root cap cells away from each other to make a tunnel like path to move through it. Injury to cells at root cap was not observed (Fig. 67). More juveniles entered through the passage made by other juveniles.
The juveniles entering the cortex did not migrate further in the inner tissue through the same path. The juveniles entering behind the root tip caused severe damage to epidermis and outer cortical cells (Fig. 68). The cells away from the nematode head were smaller but nearer to the nematode body were larger and binucleate (Fig. 69). Two to three cell layers away from nematode head, few synchronously dividing nuclei were observed (Fig. 70). The nucleoli enclosed one or more vacuoles. Occasionally some cells contained four or more nuclei (Fig. 71).
48th After Inoculation :
At this stage the cells in the provascular region exhibited hypertrophy and hyperplasia simultaneously Around the nematode head the cells of the undifferentiated phloem showed severe hypertrophy (Fig. 72). The number 154
of hypertrophied cells ranged upto five cells. These incipient giant cells contained a large central vacuole and parietally distributed granular, deeply stained cytoplasm. The average size of the giant cell measured 286x26jim enclosing upto 8 nuclei. Both the nuclei and nucleoli were hypertrophied. The nucleoli were either globuler conical, elongated, constricted, cordate or elbow shaped. Some times four to five small irregularly arranged vacuoles were observed in hypertrophied nucleoli. Giant cell were surrounded by small sized parenchymatous cells. The gaint cells were about ten times longer than small sized neighbouring cells. (Fig. 73).
72h After Inoculation:
The giant cell was now fully differentiated measuring 312x39 jjm with nematode head inside it. The granular cytoplasm became more dense and occupied almost the entire space inside the giant cell.
The size of nuclei could be related to the distance from the nematode head. The smaller the distance from the nematode head the larger the nuclei and vice-versa (Fig. 74). Smaller nuclei of 9.6^m were observed closely placed in front of nematode head. Each nucleus enclosed a small nucleolus (3.2^m). The nuclei very close to the nematode head were larger (12.8^m) and each enclosed a large nucleolus (6.4^m). similarly very small nucleoli were observed far away from nematode head.
6 Days After Inoculation:
After six days of inoculation the giant cell size increased to 155
325xl30nm. More dense and granular cytoplasm with uneven cell wall thickenings was observed (Fig. 75). Large sized nuclei in granular cytoplasm, with some nuclei possessing two-three small to large sized nucleoli were observed. Other nuclei contained only one severely hypertrophied nucleolus in the same giant cell. Synchronous nuclear divisions were observed in some giant cells. Large sized nucleus and nuclei measured 11.5^m and 4.6\im in diameter respectively. The nuclei varied in shape and were either spherical, elongate or ameoboid to oval, the vacuoles ranged between four and six in large nucleoli.
12 Days After Inoculation:
The giant cell cytoplasm bacame more dense after 12 days of inoculation due to which it became difficult to count the exact number of the nuclei. The nuclei grouped around the central vacuole. Deep stained nucleoli were quite prominent in the nuclei of giant cells. The nucleoli varied in size from a very small to very large about 6.2nm. In light stained cytoplasm of giant cell the nucleoli were easily distinguished. The nuclei varied in shape such as and globose, ovoid, elongate, triangular and even ameoboid. Upto seven nucleoli in a nucleus were found. About 10 nuclei and more than 60 nucleoli were observed in a sigle giant cell (Fig. 76)
A nematode with its tail still outside the plant body was found inducing giant cells . The giant cells surrounded by small sized compact parenchymotous cells were observed. Nuclei with distinct bounderies and 156
various shapes (elongated, amoeboid etc.) were observed. The number of nuclei in a giant cell declined and it reached up to 75 with eight nucleoli were found in the nucleus.
18 Days After Inoculation:
The giant cells reached to a maximum size of almost 530x20lum. Cell wall became more thick with increased vacuolation and reduction in number of nuclei. The number of nuclei varied from one-eight in each nucleus. The nuclei were ameoboid and nucleoli were globular, triangular or ameoboid in shape.(Fig. 77).
The giant cell remained as such with much thick cell wall. However, the cytoplasm became less dense. Towards the peripheries the giant cells were showing empty spaces. The number of nuclei reduced to about 50 with continuous reduction. Nuclei were generally ameoboid in shape and each carried ten nucleoli of various shapes, and sizes. With the increase in vacuolation the cytoplasm contents of the giant cell decreased. Though the number of nuclei decreased in the giant cells but the nucleoli like bodies increased in number in the intestine of the nematode. Most of the nematodes were metamorphising into adults after completing their fourth moult.
24 and 30 Days After Inoculation:
The size of the giant cell became constant but their contents were removed by the nematode. This cused emptying of giant cells. The nematode 157
became swollen andpyriform in shape.(Fig. 78).
After 30 days of inoculation the nematodes were found associated with egg sacs and egg masses. A few giant cells were changing into vessel like elements 30 days after inoculation.(Fig. 79, 80 & 18).
DISCUSSION
Meloidogym incognita larvae penetrated the roots of Momordica charantia at or just behind the root cap. Further migration took place intercellularly and no cellular damage occurred throughout their path. The passage formed at the tip and the occurrence of the compressed cells along the body length indicated, intercellular migration of juveniles. Penetration of the juveniles of root-knot nematodes close to the root tip and other regions of roots in tomato (Christie, 1936), anywhere from the root tip back to the region of root hair formation in sweet potato (Krusberg and Nielsen, 1958). and in the region of cell differentiation and elongation in wheat (Siddiqui and Taylor, 1970) has been reported. The observations of our studies with regard to penetration site for the juveniles are similar to the earlier reports. Inter-and intracellular migration of juveniles inside the root tissues after penetration was proposed by Nemec (1910). Studies of Christie (1936), Krusberg and Nielsen (1958) and Siddiqui and Taylor (1970) supported these observations. Later studies, however, have confirmed that juveniles of root-knot nematodes migrate intercellularly by separating the cell walls along the middle lamella, (Endo and Wergin, 1973; Jones and 158
Payne, 1978). The present observations on migration of the juveniles ofM. incognita in roots of A/, charantia support the later view.
The quick response of the inner root tissue to the nematode was the enlargement of the cells. The cell alongwith their nuclei and nucleoli hypertrophied near the head and along the body length of M incognita. Enlargement and fragmentation of the nucleoli of the affected cells occurred. Nucleolus plays an important role in the formation of ribosomes which help in protein synthesis inside the cell. The nucleoli in actively protein synthesizing cells become large than in inactive cells (Johnson and Johnson, 1986). In addition to hypertrophy, the affected cells also exhibited nuclear divisions without cell wall formation. Occurrence of 5-6 giant cells, after 48h of inoculation, indicates that the host-parasite relationship has been established during this period. The giant cells possessed upto eight nuclei and each nucleus contained one or more nucleoli. Jones and Payne (1978) also observed giant cell formation in Impatiens balsamina by M incognita, after 48h of inoculation. Siddiqui and Taylor (1970) noticed giant cells caused by M. naasi in wheat roots four days after inoculation.
Multinucleate condition of giant cell arising from repeated mitoses without cytokinesis was proposed by Huang and Maggenti (1969a). The two nuclei of a young giant cell undergo rapid, synchronous divisions without subsequent cytokinesis and upto eight nuclei can be found within 48h of nematode infection (Jones and Payne, 1978). Synchronous nuclear divisions 159
within the same giant cell have been reported by many other workers (Bird 1961; Krusberg and Nielsen, 1958; Smith and Mai, 1965, Owens and Specht, 1964; and Pasha et al., 1987). The present observations substantiate this mode of giant cell development.
After 72h and 6 days of inoculation, increase in number and size of nuclei and their change in shape from globular to ameoboid with an increase in granulation and density of cytoplasm indicated an immense metabolic activity of the growing giant cells.
Surface area of the nuclei is tremendously increased by their ameoboid shapes and irregular lobes (Huang and Maggenti, 1969a). In developing giant cells, cell organelles become abundant (Jones and Northcote, 1972; Jones and Dropkin, 1976; Jones and Gunning, 1976; Jones and Payne, 1978). Extremely dense cytoplasm, large number of nuclei and nucleoli observed on the 12th day of inoculation in the giant cell indicated increased cellular activities.
Light stained and vacuolated giant cell cytoplasm, after 12 days of inoculation, was probably due to increased feeding by the developing nematode. Root-knot nematodes feed on cytoplasm of the giant cell. Increase in vacuolation, emptying of small giant cells and decrease in number of nuclei of all the giant cells, 18 days after inoculation, further supported that increased nematode feeding caused such intracellular changes. Cytoplasmic deterioration possibly occurred due to consumption of nuclear materials by the nematodes. 160
According to Bird and Loveys (1975) and McClure (1977), Meloidogyne functions as metabolic sink in infected plants, and that is why photosynthates are mobilized from shoots to roots particularly to the giant cells. Though Meon et al., (1978) reported that mobilization and accumulation of these substances reach a maximum level when the adult females commence egg laying and decline thereafter. The present study suggests that removal of cytoplasmic material by the nematode surpasses its accumulation in the giant cells at the time of egg deposition.
When the nematode stopped feeding or died, the giant cells collapsed (Krusberg and Nielsen, (1958). From present study it becomes evident that empty giant cells do not collapse but change into abnormal vessel elements. This transformation might provide strength to the affected tissue and to prevent collapsing of galled tissues.
Thus, the juveniles oiMeloidogyne incognita penetrated at or behind the root tips oiMomordica charantia. They migrated intercellularly, in the inner tissue, by separating the cell walls. The giant cells induced in the region of unddifferentiated phloem within 48h of inoculation. Maximum number of nuclei and highly dense cytoplasm was noticed after 12 days of inoculation. Decrease in number of nuclei and increase in vacuolation was found after 18 days of inoculation. Smaller giant cells became empty and changed into vessel like elements by the deposition of lignified secondary wall material. Larger giant cells with little or no cytoplasm also transformed into abnormal vessel elements, after 30 days of inoculation. 161
B. GALL
Considerable information has been accumulated on host response to Meloidogyne spp. Host parasite relationship at the tissue and cellular levels by many workers in different plant species has been worked out. (Christie, 1936; Davis and Jenkins, 1960).
Meloidogyne spp. elecits an array of responses from its host plant. Root- knot nematode which penetrates the roots induces a conspicuous gall. Dropkin and Bonne (1966) observed gall formation by pericycle and cortical hypertrophy and hyperplasia as early as first day after inoculation. Gall formation in tomato roots was observed even when the juveniles were outside the tomato roots. (Schuster and Sullivan, 1960). Hypertrophy, and hyperplasia in cortex, pericycle and stele leading to gall formation in soybean roots were observed (Ibrahim and Massoud, 1974), cortical and stelar proliferation were observed ( Davis and Jenkins ,1960). The present work was carried out to investigate the histological changes leading to gall formation in Momordica charantia roots infected with root knot nematode Meloidogyne incognita, and to compare them with earlier findings.
OBSERVATIONS
24 h After Inoculation:
The second-stage juveniles of M incognita preferably penetrate near the root tip region. They migrate intercellularly towards the region of 162
vascular differentiation . The cells around the nematode head get hypertrophied. The enlarged cells enclosed one to four hypertrophied nuclei. Each nucleus was associated with a large vacuolated nucleolus.(Figs. 67, 68, 69, 70&71).
48 h After Inoculation:
The infected portion of the root increased in width due to galling. The increase in width was from 0.17mm to 0.39 mm in infected root. Some cells in the differentiating phloem zone were severely hypertrophied and transformed into discrete giant cells (Fig. 82) The largest giant cell measured about 275x25|im. Giant cells were surrounded by about three to four layers of small sized parenchymatous cells which were parallel to giant cells. Four to five large sized cell in between the giant cells and thedifferentiating sieve tube elements were found (Fig. 73). The vessel elements near the giant cells were wider as compared to those away from the giant cells.
72 h After Inoculation:
The average size of the gall increased and reached up to 0.51mm. However, the width of the normal roots was 0.23mm. The largest giant cell reached upto the size of 302 x 80|im. The average diameter of the xylem and cortical parenchyma ranged between 30.1 to 21.4^m respectively. Average diameter of the normal parenchyma cells was 11.7, jim. The xylem and 163
phloem parenchyma cells increased in number. Average width of the vessel elements near the giant cell was 64.0nm, but in normal root it was 28.8nm. The width of endodermal cell was 61.0[im in the galled roots and 26.7 ^im in normal roots (Fig. 83).
6 Day After Inoculation:
6 day old galled roots were 0.73mm wide as compared to normal roots which were 0.37mm in width. The giant cell reached to a maximum size of 319x 127|im with an average of 232x65^m. The average diameter of xylem and cortical parenchyma cells were 40.1 and 34.3^m respectively. The diameter of parenchyma cell in normal root was nearly about its half i.e. 19.5^m. The vessel width near the giant cell was 70.6^m and away from it was 35.3^m . In normal root it measured about 30.0nm. Endodermal cells did not show proliferation. Number of xylem and phloem parenchyma cells increased in affected than in unaffected part.(Fig. 84).
12 Days After Inoculation:
12 day old galled roots measured almost 1.48mm in width while normal root measured about 0.51mm. The largest giant cell measured about 420x155nm with an average of about 258x83^m. The xylem and cortical parenchyma cells were having average diameter of 55.1 and 46.2nm. respectively. Average diameter of xylem and cortical parenchyma cells of normal root was 21.9nm.(Fig. 76). 164
18 Days After Inoculation:
The gall measured about 2.5mm in width after 18 days of inoculation. The nematode also increase in width. The other cells did not show any increase in width or diameter.(Fig. 85).
24 and 30 Days After Inoculation:
The gall measured about 2.8 mm and 4.5 mm after 24 and 30 days of inoculation,respectively. Egg masses associated with females were observed after 30 days of inoculation. Cambial cells and preformed giant cells were found attacked by newly hatched second-stage juveniles causing secondary infection. Hyperplastic and hypertrophic reactions as a result of secondary infection were repeated. (Fig. 86).
DISCUSSION
Galling, one of the earliest host response of root-knot nematode infection, results from hyperplastic and hypertrophic reactions taking place simultaneously. Galls are induced when Meloidogyme juveniles enter the root (Christie, 1936), but they may also be induced without entry of the juveniles (Schuster and Sullivan, 1960). According to Schuster and Sullivan (1960), M incognita penetrates its stylet and secretes certain substances that stimulate host tissue to form galls. This study shows that the gall formation was induced when the juvenile reached to the region of vascular differentiation. Hypertrophied cells near the head and along the body length 165
of the nematode, within 24h of inoculation, supported the view of Schuster and Sullivan (1960).
In early stages of galling, hyperplastic and hypertrophic reactions appeared to be responsible for the gall formation in infected roots. The size of the giant cells, as a result of hypertrophy, increased ten times more than a normal cell. Presence of small sized cells near the giant cells indicates a simultaneous hyperplastic reaction in neighbouring parenchyma. In addition to giant cell formation and hyperplasia of neighbouring parenchyma cells, hyperplasia of phloem parenchyma was also found contributing in root galling, within 48h of inoculation. Vessel element dilation, near the giant cells, seems to be a hypertrophic response of root-knot nematode infection. Jones and Payne (1978) also observed giant cells and hyperplastic parenchymatous tissue within 48h of inoculation. This study is in confirmation of Jones and Payne (1978). After 72h of inoculation, xylem and phloem parenchyama cells divided actively. The main contributing factor to the gall formation was either hypertrophy or hyperplasia or both of all t>'pes of cells including vessel elements, xylem and cortical parenchyma, and also endodermal cells. Hypertrophy of giant cells was found until 18th day of inoculation whereas increase in size of xylem and cortical parenchyma was observed 12 and 18 days after inoculation, respectively. The amount of xylem and phloem parenchyma increased upto 12 days of inoculation. Thus, hyperplastic and hypertrophic reactions of different types of cells, in the affected region, led to the formation of galls until 15th day of inoculation. 166
The size of the nematode incrased slowly upto 18 days after inoculation, but it increased rapidly after 18 days. Increase in gall size, after 18 days of inoculation, was due to nematode development into adult female. The adult female attained its maximum size after 24 days of inoculation. Production of egg masses by the adult females further contributed in increasing the gall size.
Secondary infection was noticed after 30 days of inoculation. The egg masses of all the females were not expelled out of the plant tissue. Some egg masses remained inside. Eggs of these egg masses hatched and the second-stage juveniles, traversing intercellularly through cortical cells, reached the cambial zone and caused secondary infection. Repetition of hypertrophic and hyperplastic reactions, due to secondary infection, caused a rapid increase in gall size. Some freshly hatched second-stage juveniles were found associaated with preformed gaint cells. These juveniles, instead of inducing new giant cells, started feeding on old giant cells. Hypertrophic and hyperplastic reactions were accompanied with such type of feeding.
Gall formation due to (i) production of giant cells, (ii) hyperplasia of pericycle and xylem parenchyma, (iii) hypertrophy of cortex, pericycle, xylem parenchyma, and metaxylem, (iv) enlargement of the nematode body and (v) production of egg masses has been reported by Christie. (1936); Dropkin (1954); Krusberg and Nielsen, (1958); Davis and Jenkins, (1960); Dropkin and Nelson, (1960); Bird, (1961; 1962); Owens and Specht, 167
(1964); Hodges and Taylor, (1966); Siddiqui and Taylor, (1970); Siddiqui, (1971a); Orr and Morey, (1978); Jones and Payne, (1978); Hisamuddin (1992). From this study it may be concluded that gall formation is also influenced by hpertrophy of endodermal cells, and hyperplasia and hypertrophy of phloem elements, in addition to other factors listed above. Rapid increase in gall size, after 30 days of inoculation, results from secondary infection.
C. VASCULAR ELEMENTS
Meloidogyne spp. infection results in the pathological development of roots and disruption of both xylem and phloem strands. Christie (1936) reported that xylem often failed to develop at the infection site, and that short irregular, unorganized xylem elements often formed from parenchyma cells in older galls, Abnormal vessel elements of irregular shapes and sizes were derived from xylem parenchyma (Davis and Jenkins, 1960; Siddiqui and Taylor, 1970). Swamy and Krishnamurthy, (1971) observed initial infection in primary phloem in young roots and in secondary phloem in older roots or ray tissue. They also reported that functional phloem after infection was destroyed and secondary phloem was not differentiated. Siddiqui and Ghouse (1975) on the other hand, observed differentiation of secondary phloem which differed from normal phloem. Byrne et. al., (1977) reported primary phloem or adjacent parenchyma to be the initial target of M. incognita juveniles. The following studies were carried out(l) to observe abnormalities in orientation, structure and origin of vessel and phloem 168
element after Meloidogyne incognita infection in Momordica charantia roots, (II) to investigate any link between giant cells and phloem and (III) to find out whether xylem and phloem strands are disrupted in infected root tissue.
OBSERVATIONS
a. XYLEM
24h After Inoculation:
The second-stage juveniles oi Meloidogyne ;>?cog«/7a penetrated the young roots of Momordica charantia at or near the root tip region and migrated to the region of vascular differentiation. Longitidunal sections of \0\i thickness did not reveal fully differentiated protoxylem strands near the incipient giant cells. However, differentiation of protoxylem elements with their spiral thicknenings of secondary wall deposition was times observed (Fig. 67, 68, 69, 70 & 71).
48h After Inoculation:
The giant cells became prominent and were differentiated fully. Near the giant cells there were hypertrophied and hyperplastied parenchymatous cells. The protoxylem strands were curved instead of straight when seen in longitudinal section towards the giant cells (Fig. 72, 73, 82).
72h After Inoculation:
The lengths of vessel elements of metaxylem strands gradually 169
decreased and width gradually increased towards the giant cells. Vessel like elements arising from parenchyma cells were observed for the first time. Some parenchymatous cells bordering the giant cells transformed into abnormal vessel elements Secondary wall depositions of these elements were typically reticulate.(Fig. 74, 83).
6 Days After Inculation:
Near the giant cells the vessel elements became more broader than longer. As seen in longitudinal section the xylem strands appeared broken abruptly or in patches near the giant cells. Some abnormal vessel elements arising from small as well as large hypertrophied parenchyma cells were observed. The shape and size of the newly formed abnormal vessel elements corresponded to the parenchyma cells from which they were originated. Addition of these vessel elements was not in any order direction but in a zig-zag manner. All the the abnormal vessel elements were showing reticulate thickenings (Fig. 75, 84).
12 Days After Inoculation:
The number of abnormal vessel elements increased progressively. The parenchyma cells around the giant cell complex transformed into vessel elements which covered the giant cell cluster. At the same time the parenchyma lying in between the normal xylem strand and abnormal vessel elements around the giant cells also changed intovessel like elements. (Fig. 76). 170
18, 24, and 30 Days After Inoculation:
Progressive increase in abnormal vessel element formation was observed 18 to30 days after inoculation. However, after 30 days of inoculation some large empty or almost empty giant cells were also found transforming into vessel like elements.(Fig. 77, 78, 79, 80 & 81).
DISCUSSION
In this experiment abnormality in orientation of xylem strands was observed 48h after inoculation where they were pushed away from their normal position due to proliferation of giant cells, and due to hyperplasia and hypertrophy of neighbouring parenchyma cells. This abnormal orientation became more pronounced 6 days after inoculation when metaxylem strand was found traversing from one side to the other in a zig-zag manner, when seen in longitudinal section. After 12 days of inoculation the metaxylem strands were also seen as irregularly scattered patches. But whatever be the orientation of xylem strands, their continuity was always maintained throughout the gall. Christie (1936) was of the opinion that vascular strands passing around the giant cells were pushed out of the normal position. Continuity of vascular cylinder, however, reamined undisturbed. Occurrence of xylem and phloem in scatterred patches was observed throughout the gall. Interruption in their continuity due to giant cell formation has also been reported (Davis and Jenkins, 1960; Odihirin and Jenkins, 1965). The present study supports the former view. 171
Abnormality in structure of normal xylem elements was noticed 48h after inoculation. The width of metaxylem elements in affected part increased two times than in unaffected part. After 72h of inoculation, vessel length was decreased to less than half in affected part. A few elements became more broader than longer. These abnormalities might be due to certain stimuli responsible for hypertrophy of different type of cells. After 6 days of inoculation, metaxylem elements not only shortened and proliferated but also deformed and assumed irregular shapes. After 12 days of inoculation some vessel elements were showing projections. In this way, normal vessel elements changed into abnormal vessel elements due to change in their structures. Hypertrophy in vessel elements was reportedby Siddiqui and Taylor (1970). This study supports the view of hypertrophy of vascular strands.
Abnormality in xylem due to origin, from cells other than procambium was evidenced 72h after inoculation. These were enclosing nuclei but not cytoplasm. And, the other type originated, 18 days after inoculation, from the empty giant cells. Formation of abnormal vessel elements from parenchyma in Meloidogyn spp. induced galls has been reported in all the host plants (Christie 1936; Krushberg and Nielsen, 1958; Davis and Jenkins, 1960; Odihirin and Jenkins, 1965; Eversmeyer and Dickerson, 1966; Siddiqui and Taylor, 1970; Siddiqui, 1971a, 1971b; Swamy and Krishnamurthy, 1971; Farooq, 1973; Siddiqui et al., 1974; Ngundo and Taylor, 1975; Ediz and Dickerson, 1976; Jones and Dropkin 1976; Byrne etal., 1977;Finley, 1981; 172
Jones, 1981; Pasha et al., 1987; Hisamuddin 1992; Hisamuddin and Siddiqui, 1992a). The present study also supports the transformation of parenchyma cells into vessel elements. In addition to parenchyma, giant cell transformation into abnormal vessel elements has also been investigated through this study. The abnormal vessel elements can, thus, be grouped, on the basis of their size, into small, large and giant vessel elements.
Growth of root tips is temporarily checked which results in poor nutrient uptake, when roots are invaded by Meloidogym juveniles (Hussey, 1985). To compenesate the loss of water and nutrients, caused by root growth inhibition, probably, lateral root branching is stimulated as has been reported by Christie (1936); Krusberg and Nielsen (1958); Davis and Jenkins (1960). It is supposed that large number of lateral branches facilitate absorption of water and mineral nutrients in larger amounts.
Although lateral root branches absorb nutrients in sufficient amount but these are not translocated to the shoots due to unsteady upward translocation (Oteifa and Elgindi, 1962; Hanowanik and Osborne (1975). To surmount the unsteady or impaired translocation, probably, plant adapts itself to produce xylem elements in large amounts. Thus, it is suggested that the plant attempts to overcome the loss of water due to disordered orientation of vessels partly by adaptation of vessels to have wide lumen and partly by the production of enormous amount of abnormal xylem.
Abnormal xylem may also function to interconnect giant cells with 173
xylem strands. Giant cells, produced in protophloem, are highly metabolically active cells and function as sinks for metabolites. They may need rapid supply of water for their unceasing metabolic activities. Since water can not flow rapidly through parenchyma, therefore, the parenchyma cells in between the giant cells and the xylem strands are transformed into vessel like elements.
Another function of abnormal xylem might be the protection and the support. The giant cells which are highly specialized cells are covered by abnormal vessel elements which transform from neighbouring parenchyma cells. In this way the giant cells are given protection to prevent them from collapse. According to Knisberg and Nielsen, (1958) if mature nematode stops feeding or dies, the giant cell cytoplasm degenerates and disappears, and finally the giant cell is collapsed. This study shows that giant cells instead of being collapsed are transformed into stronger cells i.e. vessel elements. The giant cells transformed into vessel like elements may or may not have cytoplasm. This function again seems to provide mechanical support to the giant cell, and to prevent the entire gall from being collapsed.
From this study it may be concluded that the abnormality in orientation of vascular strands was started 48h after inoculation. Vascular strands were severely distorted due to the multiple hypertrophic and hyperplastic reactions taking place continuously. The strands became wavy and appeared as scattered patches, when seen in longitudinal sections. The vessel elements become 174
structurally abnormal within 48h of inoculation. The vessel elements broadened near giant cells due to hypertrophic reactions. Irregular shape and size of vessel elements of metaxylem strands were consequenced upon pressure exerted on them by the tissue resulting from hypertrophic and hyperplastic reaction. Abnormal origin of vessel elements was observed 72h after inoculation from small parenchyma cells; 12 days after inoculation from hypertrophied parenchyma cells; 18 days after inoculation from small giant cells; and 30 days after inoculation from larger giant cells. From these observations, it is suggested that formation of vessel elements in excess amount might increase upward translocation, or supply water to giant cells, or provide protection to giant cells, or give support to entire gall to prevent its collapse.
b. PHLOEM
24h After Inoculation:
After penetration, most of the juveniles oi Meloidogyne incognita were found only in the zone of differentiation of the growing root. However, the juveniles which reached the differentiation zone having their head in the protophloem when seen in longitudinal section In transverse sections their bodies were found only in the protophloem region(Fig. 67, 68, 68, 70 & 71).
48h After Inoculation:
After 48h of inoculation some of the cells near the nematode head 175
changed into discrete giant cells. These giant cells were having dense cytoplasm and many nuclei. There were few cells of the same diameter in between the giant cells nd sieve tube elements of protophloem when seen in longitudinal section.(Fig. 72, 73, 82).
72h After Inoculation:
The giant cell size increased. The number of cells in between the giant cells and the sieve tube elements of the phloem also increased.(Fig. 74, 83).
6 Days After Inoculation:
The giant cell size and density of cytoplasm further increased after 6 days of inoculation. Phloem strand running near the giant cells were observed in the longitudinal section of the gall. In some sections the phloem strands appeared as if ending abruptly at the giant cell. At such instances a group of parenchyma cells with few sieve tube elements was found at the junction of the giant cell and the phloem strands. Althogh the shape and the size of these sieve tube elements corresponded to the surrounding cells but they could only be identified on the basis of the presence of slime plugs. The sieve ube elements of the phloem traversing by the side or around the giant cell were not deformed (Fig. 75, 84).
12 Days After Inoculation:
The giant cells were observed for the first time in cortical region. 176
The consecutive serial sections revealed that cortical giant cells were associated with the vascular strands of rootlets. The giant cells were associated with sieve tube elements. (Fig. 87). Phloem strands appeared broken.It seemed to be ending at one giant cell and emerging from another.
18 Days After Inocuation:
The orientation of phloem cells was very much disturbed with the increase in size of nematode body. The sieve tube elements were now more elongated. Most of the cells of protophloem were crushed or were pushed on either side of the nematode body. The secondarily developed phloem tissue comprised of both long as well as short cells.(Fig. 77, 78).
24 and 30 Days After Inoculation:
The development of nematode was complete. Abnormal xylem appeared completely entangling the giant cell cluster when seen in longitudinal sections. When seen in tranverse sections the outer most giant cell of a cluster was found connected with phloem. The phloem elements were irregularly arranged and lacked a definite pattern. Abrupt changes in phloem elements and consequntly in their dimensions were observed. In transverse sections, the sieve tube elements were seen alternately arranged in tangential and radial patches near the giant cells.
After 30 days of inoculation no further changes were observed, the main feature was the occurrence of newly hatched second stage-juveniles 177
which either induced fresh giant cells or fed on old ones (Fig. 79, 80 & 81).
DISCUSSION
Hypertrophy of cells near the nematode head, although, was observed within 24 h of inoculation but discrete giant cells were observed only after 48 h of inocualtion. The preferential feeding site of the nematodes appeared to be only the phloem region. The nematodes which did not induce giant cells in undifferentiated zone were found lying with their heads in phloem region, as was observed 24h after inoculation. Presence of parenchyma like cells in between the giant cells and the sieve tube elements indicated that nematode induced hyperplasia in undifferentiated cells which were to be transformed into sieve tube elements. Ediz and Dickerson (1976) also found most of the giant cells in phloem region. Primary phloem or adjacent parenchyma were selected as feeding sites by nearly all the root-knot nematodes (Byrne et aL, 1977; Finley, 1981).
Since giant cells are highly metabolically active cells, therefore, they should directly or indirectly be connected with the phloem. This communication is essential for a continuous supply of assimilates to carry out cellular activites at its required rate. This view is strengthened by observing sieve tube elements ending abruptly on the walls of the giant cells after 6 days of inoculation. Formation of sieve tube elements from the hyperplastied parenchymatous cells of the phloem further support that giant cells are connected with the phloem. 178
After 12th days of inoculation, phloem seems to be the most affected tissue. At this stage orientation of sieve tube elements was disturbed firstly due to giant cell formation and secondly due to nematode development. Because of these two factors the phloem strands were dispersed.The sieve tube elementSjinstead of traversing in a straight column, were passing to the left, right, above and below the nematode and the giant cells. Continuity of phloem strand, however, was not broken. Discontinuity between sieve tube elements had been observed by Jacobs and Marrow (1958) in early stages of phloem development, after infection.
Sieve tube elements of irregular shape and size, observed after 12 days of inoculation near the giant cells, were formed probably due to two reasons. Firstly because of hypertrophic reaction, and secondly due to transformation of sieve tube elements from phloem parenchyma cells. The juveniles that penetrated later, probably, caused giant cell formation in the cortex. They attacked undifferentiated vascular strands of the root-lets. Formation of giant cells in the cortex and their derivation from cortical parenchyma have been reported in many plants (Krusberg and Nielsen, 1958; Ediz and Dickerson, 1976). This observation supports that giant cells were not derived from cortical parenchyma but from undifferentiated meristematic cells. The giant cells observed in the cortex were not independent of the phloem, but they were always connected with the phloem strand of the root- lets. Thus, the root-lets produced in response to root- 179
knot nematode infection (Christie, 1936; Kusberg and Nielsen, 1958) and supposed to increase absorption of water, also provide more number of vulnerable sites for nematode attack. Orientation, shape and size of sieve tube elements were very much affected after 18 days of inoculation. Protophloem was almost completely crushed mainly due to nematode development. Orientation and shape of sieve tube elements were also changed due to the enlargement of the nematode body.
The giant cells more or less surrounded by the xylem were reported by Christie (1936), Krusberg and Nielsen (1958). They did not mention any other tissue adjacent to the giant cells except the xylem. Siddiqui and Taylor (1970) found giant cells, completely surrounded by xylem after 12 days of inoculation, Finley (1981) stated that the giant cells were formed in the phloem tissue of roots, stolons, and tubers of potato as a result of Meloidogyne chitwoodi infection. Completely suppressed phloem was reported by Swamy and Krishnamurthy (1971) in Meloidogyne incognita infected Basella roots. Abnormal sieve tube elements with unusual orientations were formed in Lagenaria roots after the destruction of primary phloem as a result of M.javanica infection (Siddiqui and Ghouse, 1975).
From our study it may be concluded that, although giant cells appeared completely enclosed by abnormal xylem elements, but the serial section study revealed that none of the giant cell was completely enveloped by the 180
xylem. The giant cells were always connected with the phloem. The sieve tube elements as seen in transverse section, instead of forming a complete ring, appeared diverting towards the giant cells. In this ways the supply of assimilates to the giant cells was not disrupted. 181
Table -1 : Tertiary bulyl alcohol dehydration schedule (Johansen, 1940)
Quantity (ml) needed for solution
Step % Alcohol Time Distilled 95% 100% 100% water ethanol ethanol T.B.A
1 50 2h or more 50 40 0 10
2 70 over night 30 50 0 20
^ J 85 1-2 h 15 50 0 35
4 95 1-2 h 0 45 0 50
5* 100 1-3 h 0 0 25 75
6 100 1-3 h 0 0 0 100
7 100 1-3 h 0 0 0 100
8 100 over night 0 0 0 100
* TBA Changes were carried out at 30°C 182
Table - 2 : Safranan and fast-green schedule (Sass, 1951)
^Step Solution Time "^
1 Xyloie 5niin
2 absduteethanol Smin
3 95%ethanol 5min
4 70%ethanol 5min
5 50%ethanol 5min
6 30%ethanol 5min
7 1% aqueous safranin 0 l-12h
8 rinse in tap water
9 30%ethanol 3min
10 50%ethanol 3min
11 70%ethanol 3min
12 95%ethanol 3min
13 0 1% fast green FCF in 95% ethanol 5-30 sec
14 absolute ethanol 15 sec
15 absolute ethanol 3min
16 xylene-absolute ethanol (11) Smin
17 xylene 5min
18 xyleiK 5 min or longer 183 a 2L
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"- ? u 3 o ••i* B '«* es .4> E 00 Cv C C c <«^ «^ § ^ oc r- o o OC Tf — — «ri ^ o _SC _f^ <,~N •3 V a> 0^ JZ ^N c O f - "^ ?? = = H 'K f a g •/^ ON f*- »ri r^ TJ- c a.re I LIST OF FIGURES Experiment 1 Figs. 1-17 Experiment 2 Figs. 18-31 Experiments Figs. 32-46 Experiment 4 Figs. 47-66 Experiments Figs. 67-87 ABBREVATIONS AP Abnormal phloem AVE Abnormal vessel element AX Abnormal xylem C Conidial chain CT Conjuctive tissue E Eggs EM Egg mass Ep Epidermis ES Eggsac GC Giant cell GCC Giant cell complex H Hyphae J Juvenile N Nematode n Nuclei na Necrotic area NH Nematode head nu Nucleoli NP Normal phloem Pa Parenchyma Ph Phloem strand VE Vessel element FIGURES Experiment 1. Fig. 1 : Showing normal root of Momordica charantia in transverse section no X). Fig. 2 : Showing heavy infestation of Meloidogyne incognita (N). The mature females have egg masses (EM). Abnormal xylem (AX) and abnormal phloem (AP) are in abundance. (25 X) Fig. 3 : Showing normal xylem (NX) strands with hyphae (H) of Paecilomyces lilacinns traversing through the lumen of vessel element. (32 X) Fig. 4 : Showing normal xylem (NX) strands with conidiophores and conidial chain (C) in the lumen of vessel element. (31.25 X) Fig. 5 : Showing hyphae (H) and conidial chains (C) in and around the nematode (N) : Hyphae destroyed eggs (E) and egg masses. (40 X) Fig. 6 : Showing abundant growth of P. lilacinus hyphae (H) in and around the female of M. incognita (N). (32 X) rx^ .?, :h ^\ ^ a^r *5 i& -*- H 6 ^-^ . M A^ Fig. 7 . Showing hyphae (H) and conidial chains (C) at the root surface. (12.5 X) Fig. 8 : Showing destroyed eggs (E) and egg masses (EM) by the fungal hyphae(H). (20X). Fig. 9 : Showing vessel elements (VE) in transverse section enclosing conidial chains (C). (40 X) Fig. 10 : Showing hyphae (H) in giant cell (GC) and nematode (N) adjacent to the giant cell. (25 X) Fig. 11 : Showing profuse fungal (H) growth around the nematode (N). (20 X) Fig. 12 Showing growth of fungal hyphae (H) inter - and intracellularly. (31.25 X) 7%^ immi Fig. 13 Showing destruction of eggs (E) and egg masses by the fungal hyphae(H). (25X) Fig. 14 : Showing hyphae (H) and conidial chains (C) in abnormal vessel element (AVE). (31.25 X) Fig 15: Showinggiant cells (GC) around the head of the nematode (NH). (15.62 X) Fig. 16 : Showing fungal hyphae (H) with conidial chians (C) around the nematode (N). (31.25 X) Fig. 17. Showing hyphae and conidial chains (C) in the lumen of abnormal vessel element (AVE). (31.25 X) Experiment 2. Fig. 18 : Showing mature female (N) with egg mass (EM) and abnormal xylem(AX) (3.2 X). Fig. 19 : Showing giant cells (GC), Abnormal xylem (AX), abnormal phloem (AP), a mature female (N) with an egg mass (EM). (10 X) Fig. 20 : Showing giant cells (G.C.) with dense cytophlasm containing multinucleolate (nu) nuclei (n). Also shows phloem strands (Ph) connected to gaint cells. (20 X) Fig. 21 ; Showing a developing nematode (N) causing giant cell (GC) formation near its head. Abnormal xylem (AX) and abnormal phloem (AP) near the giant cells is prominent. (25 X). Fig. 22 : Showing unaffected root in T.S., having normal xylem (NX) and parenchymatous conjuctive tissue (CT). (4 X) Fig. 23 : Showing normal xylem (NX) and normal phloem (NP) as seen in L.S.(IOX) Fig. 24 : Showing giant cell (GC) abnormal xylem (AX) abnormal phloem (AP), a mature nematode (N,) with egg mass, and developing nematodes (N,, N3 and NJ. (10 X) Fig. 25 : Showing normal xylem (NX) and normal parenchyma (Pa). (20 X) Fig. 26 : Showing mature females (N) giant cell complexes (GCC), abnormal xylem (AX) and abnormal phloem (AP). (3.2 X) Fig. 27 : Showing mature females in cortex (N,), conjunctive tissue (N,) and xylem arches (NJ. (2.56 X) Fig. 28 : Showing giant cell complexes (GCC) abnormalities in xylem, phloem and other tissues as seen in T.S. (3.2 X) Fig. 29 : Showing abnormalities in xylem, phloem and other tissue as seen in L.S. The mature feamle with its egg mass is also prominent (4X) Fig. 30 : Showing giant cell complexes (GCC) and mature females (N). (10 X) ^ / T •*»>*«r' ^^^M ^ \ %c:^. 4 *' ^s^ -t Fig. 31 : Showing giant cells (GC) containing dense cytoplasm, large sized nuclei (n) and abnormal xylem (AX). (10 X) Experiment 3. Fig. 32 : Showing two females (N) and more than ten giant cells (GC). (15.62 X) Fig. 33 : Showing one female (N) in medullary ray in transverse section. (10 X) Fig. 34 : Showing three mature females (N) with almost empty giant cells (GC). (3.2X) Fig. 35 : Showing giant cells (GC) with dense and granular cytoplasm at lower inoculum level. Nuclei (n) with large nucleoli (nu) prominent. (10 X) Fig. 36 : Showing two females (N) and less granular giant cell cytoplasm (GC). (3.2X) Fig. 37 ; Showing large giant cells (GC) with less giant cell cytoplasm (GCG) at higher inoculum level. (10 X) Fig. 38 : Showing empty, small sized giant cells (GC) at lower inoculum level. (15.62 X) Fig. 39 : Showin medium sized giant cells (GC) with little cytoplasm and, large sized giant cells with more cytoplasm at lower inoculum level. (10 X) Fig. 40 : Showing a giant cell (GC) with nuclei (n) enclosing four nucleoli (nu). (40 X) Fig. 41 : Showing a mature female (N) with an egg sac (ES) containing eggs. (10 X) Fig. 42 : Showing a mature female (N) without an egg sac. (10 X) GC SJ."^ >* ' * Fig. 43 : Showing more amount of abnormal xylem (AX) at higher inoculum level. (12.5 X) Fig. 44 : Showing less amount of abnormal xylem (AX) at lower inoculum level. (4 X) Fig. 45 : Showing much distortion in the normal xylem (NX) strand at higher inoculum level. (12.5 X) Fig. 46 : Showing less distortion in the normal xylem (NX) and normal phloem (NP) strands at lower inoculum level. (12.5 X) Experiment 4. Fig. 47 : Showing galling in root having no giant cells. (4 X) Fig. 48 : Showing less amount of abnormal xylem (AX) and a developing nematode (N). (25 X) W^'3^t -fct. i/n ._^^ A ?i'.S v"^' ^^- Fig. 49 : Showing less disruption in vascular tissues (in transverse section). (25 X) Fig. 50 : Showing necrotic area (na) in the cortical tissues. (12.5 X) Fig. 51 : Showing developing female (N) and small sized giant cells (GC). (12.5 X) Fig. 52 : Showing one female (N) but less distortion in vascular strand. (20 X) Fig. 53 : Showing one female (N) and small sized giant cells (GC). (20 X) Fig. 54 : Showing egg mass (EM) associated with one female (N). (20 X) %r' Fig 55: Showing nematodes (N) and giant cells (GC) in transverse section. (2.56 X) Fig 56 : Showing giant cells (GC) with dense and granular cytoplasm. (12.5 X) Fig. 57 : Showing much disruption in the root in T.S. (2.56 X) Fig. 58 : Showing few nematodes (N) but less disruption in vascular tissues. (2.56 X) Fig. 59 : Showing many nematodes (N), giant cells (GC) surounded by abnormal xylem (AX). (2.56 X) Fig. 60 : Showing females (N) in T.S. (3.2 X) t. i5tr^^*^^>9 . Fig. 61 : Showing many nematodes (N), abnormal xylem (AX) and giant cells (GC). (3.2 X) Fig. 62 : Showing mature and developing females (N) causing heavy damage to the root. (10 X) Fig. 63 : Showing females (N) and egg masses (EM). (3.2 X) Fig. 64 : Showing giant cells (GC) with dense and granular cytoplasm. (12.5 X) Fig. 65 : Showing more females (N) with more abnormalities in the root tissues. (4.0 X) Fig. 66 : Showing feamles (N) in all parts of the root. (3.2 X) Experiment 5. Fig. 67 : Showing second-stage juveniles (J) in root tip, migrating to zone of differentiation. (20 X) Fig. 68 : Showing peeling off of epidermis (Ep) by the second - stage juveniles (J). (10 X) Fig. 69 : Showing hypertrophied cells with large nuclei (n) near the nematode head (Nh). (25 X) Fig. 70 : Showing dividing nuclei (n) in presence of second-stage juveniles (J). (20 X) Fig. 71 : Showing hypertrophied cell with four nuclei (n). (20 X) Fig. 72 : Showing hypertrophied cell near the head of second-stage juvenile (J). (20 X) Fig. 73 : Showing incipient giant cell (GC) near the head of second stage juvenile (J). (20 X) Fig. 74 : Showing large sized nucleus (n) near the nematode (N) head. (31.25 X) Fig. 75 : Showing incipient giant cell (GC) with uneven thick cell wall, dense cytoplasm and a large nucleus (n) near the nematode body (N). (31.25 X) Fig. 76 : Showing giant cell (GC) with dense cytoplasm and many large nuclei (n) and a developing nematode (N). (25 X) Fig. 77 : Showing giant cells containing variously shaped nuclei (n) near the nematode (N). (15.62 X) Fig. 78 : Showing a large giant cell (GC) with dense cytoplasm and prominent nuclei and (1.2,3) small empty giant cells. (15.62 X) Fig. 79 : Showing eggs (E) emerging out of egg sac (ES). (10 X) Fig. 80 : Showing a mature female (N) and giant cell (GC). (15.62 X) Fig. 81 : Showing a giant cell (GC) changing in abnormal vessel element (AVE). (20 X) Fig. 82 : Showing hypertrophied incipient giant cell (GC) in the phloem region. (32 X) Fig. 83 : Showing giant cell (GC) and other hypertrophied cells. (25 X) Fig. 84 : Showing hyperplasia in parenchyma (Pa) near the giant cells (GC) and the nematodes (N). (15.62 X) Fig. 85 : Showing a developing nematode (N) near the giant cells (GC). (25 X) Fig. 86 : Showing second-stage juveniles (J) causing secondary infection. (10 X) Fig. 87 : Showing phloem strands (Ph) ending at giant cell (GC). (25 X) 'I* 87 SUMMARY Momordica charantia is an important cucurbit vegetable crop grown in almost all parts of India. The root-knot nematode, Meloidogyne incognita causes considerable losses to this crop. Following studies were carried out to investigate the mechanism of disease incidence, the effects of nematode infection on the plant growth, and to observe the various developmental stages of the nematode before and after infection. Meloidogyne incognita, soon after penetration, induced giant cell formation, hyperplasia and hypertrophy around the giant cells. These changes led to the formation of prominent galls. As a result of infection, the plant growth retarded and the plants exhibited stunting and loss of weight. The harmful effects of infection on physiological activities of the plants resulted in yield loss. The experiments were designed to study the effects of disease on plants and to check the disease as far as possible. Two methods of disease control were selected to limit heavy economic losses. In one experiment, a biocontrol agent Paecilomyces lilacinus was used as control measure. The fungus was added into the soil around the roots before and after of nematode inoculation. The fungus was applied at specific time period after seedling transplantation. It's effect on the plant growth, nematode development and secondary infection was observed 194 Paecilomyces lilacinus is an effective biocontrol agent to check root-knot nematode infection. The fungus P. lilacinus promoted growth of root-knot nematode infected plants. The lengths and weights of roots and shoots of Meloidogyne incognita infected and P. lilacinus treated plants enhanced as compared to untreated plants. Leaf area, number of branches and flowers of P. lilacinus treated plants increased. Number and size of the gall reduced due to the presence of the fungus. The egg m^ses^ were destroyed by the fungus. The most effective time of fungus application was one week before nematode inoculation. The fungus not only developed in the soil and around the root surface but also entered into the root tissues without damaging the plarit. In all the treatments, P. lilacinus was found destroying eggs and egg masses of root-knot nematode. The efficacy of the fungus declined as the time of application increased. In all the treatments it was found that the fungus destroying egg masses checked secondary infection. In fungus treated plants, the galled roots had smaller amount of abnormal xylem and phloem as compared to untreated plants. The giant cells were smaller and contained less cytoplasm. In the second experiment aldicarb, a non fumigant nematicide was used to control the root knot disease and to improve the plant growth. The nematicide was applied at the time of inoculation, and then after one to four weeks after inoculation. 195 Aldicarb, a systemic nematode, was used in varying concentrations and at varying intervals of time, after nematode inoculation, to see the effect on the nematode development, plant growth, and on the tissues in galled regions of the root. As compared to control, the lengths of roots and shoots, fresh and dry weights of roots and shoots decreased drastically in nematode incoulated and untreated plants. The lengths and weights of roots and shoots increased in nematode inoculated and simultaneously aldicarb treated plants. In comparison to nematode inoculated plants, the lengths and weights of alidcarb treated plants were higher. However, a decreasing trend was observed from T3 to T, among aldicarb treated plants. In all the aldicarb treated plants the values of plant grwoth parameters were higher than T^ plants. Number of branches per plant, leaf area, and number of flowers were highest in control plants followed by T^, T^, Tj, T^and Implants. In T^ plants the readings were lower than other treatments. The size and the number of galls, and the number of egg masses were highest in T^ plants and lowest in T3 plants which exhibited increasing trend from the treatments T3 to T,. The galls of the treatment T^ contained more abnormal xylem and phloem than T, plants. Amount of abnormal vascular tissues increased from T3 to T^ treatments. Among the aldicarb treated plants the most effective time of controlling the disease was thatjof applying aldicarb simultaneously and less effective when applied after one or more weeks of nematode inoculation. The efficacy of aldicarb gradually decreased as the time interval increased from one week to four weeks after inoculation. In all the 196 treatments, it was observed that secondary infection was checked probably because of residual effects of aldicarb. Simultaneous application of aldicarb was most effective that might have killed the juveniles before penetration but later applications did not kill the juveniles that had entered roots but checked their development within the roots. The third experiment was aimed at knowing the effect of different inoculum levels of the nematode on the growth of the plant, on the formation of the galls, on the development of the nematodes, and on the formation of abnormal tissues inside the galls. An initial population density produces different effects on different host plants. Low population level may not affect a plant, or may be beneficial or harmful. Momordica charantia responded differently to different population densities. At the lowest inoculum level there was not any remarkable decline in growth of plant as compared to control. At 50 J^, a slight but non-significant increase was observed. At higher initial inoculum levels the growth decreased significantly. Reduction in lengths and weights of both roots and shoots was maximum at 5,000 J^, the highest inoculum level. The galls were scanty and very small at lowest (Pi=5 J^) initial inoculum level. The gall number and the gall size increased from lower to higher inoculum levels with the maximum at highest inoculum level. The number of mature females recovered from plants at Pi = 05 l^. increased to 197 maximum at Pi = 5,000 Jj. However,, their size decreased as the inoculum level increased. At lowest inoculum level one nematode was enough to cause the formation of giant cell complex. While at higher inoculum levels more nematodes were found causing multiple giant cell complexes. The average size of giant cell was large at lower Pi and small at higher inoculum level. The giant cell cytoplasm was more dense at lower inoculum level than at higher inoculum level. Abnormalities in the orientation and structure of xylem and phloem were few at lower initial inoculum level and more at higher inoculum levels. At lowest Pi the number of giant cells around the head of mature female were 6 to 8. The gaint cells were larger in size enclosed dense cytoplasm as compared to those found in the plant at high Pi. The amount of abnormal xylem and phloem was more at high Pi than at lower Pi. Out of four primary inoculum levels (OSJ^, SOJj, 500 J^.and 5,000 J^) considered, the galling was scanty and also the size of the gall was very small at the lowest inoculum level. The number of mature females recorded was small, at lowest inoculum level however, their size was large as compared to the other inoculum levels. At this level the number of egg masses obtained was large. By the increase in primary inoculum level, the plant growth gradually reduced, number and size of the gall increased, number of mature females 198 per gram root increased but the size of the mature female decreased. The number of egg masses per plant, number of eggs per eggmass also increased. Greatest reduction in length and weight of plant was observed at the highest initial inoculum level. The number and the size of the gall, the number of mature females per gram root and the number of egg masses per plant were maximum at the highest inoculum level. The size of the mature female and number of eggs per eggmass decreased at higher inoculum levels. In another experiment different varieties of bitter go urd available in the market were examined to find out any Meloidogyne incognita resistant variety. This experiment was conducted because previously tested resistant variety is not available. The six varieties viz Faizabadi, Jhalarwali, PDM, Jaunpuri, Baramasi and Aligarh local obtained from different seed sources were evaluated for this purpose. Out of the six different varieties oi Momordica charanlia selected, Aligarh local and Baramasi exhibited highest reduction in length and weight at various initial inoculum levels, as compared to control. The number and size of the galls and the number of the mature females were more in these two varieties. The variety Faizabadi exhibited least effect oi Meloidogyne incognita on plant growth. The gall number was lowest in this variety. In Jhalarwali variety the reduction in plant growth was more than Faizabadi but lower than other four varieties. It produced more galls than Faizabadi. The plant growth of nematode infected PDM and Jaunpuri plants was lower than Faizabadi and Jhalarwali but higher than Baramasi and Aligarh Local. 199 In the variety Faizabadi no galling was observed and the galls ii produced were small. In these galls, the giant cells were small, and contained little cytoplasm. The nematode either died or did not reach to mature stage. The galled regions were not distorted. Abnormal xylem and phloem was scarce. In other varieties number and size of galls, size of giant cells increased, being maximum in Aligarh local followed by Baramasi, Jaunpuri, PDM and Jhalarwali. In the last experiment Meloidogyne incognita infected roots were examined from the day one to the 30th day, after inoculation. At regular intervals of time anatomical studies were carried out to investigate sequential changes in the formation of giant cells, in the development of the nematode, and in the formation of hypertrophic and hyperplastic tissue and abnormal vascular elements. The characteric feature in Meloidogyne induced galls is the formation of discrete, abnormally large giant cells. In growing roots, the juveniles of M. incognita induced giant cell in provascular elements specially from the cells which develop into primary phloem. Soon after penetration, the juveniles entered the procambium zone and incited hypertrophy and hyperplasia not only in sieve tube transforming cells but also in the nearby cells. A large number of cells around the nematode head enlarged and many of them divided leading to the formation of the gall. The affected cells comprised of the cells of cortex, endodermis, pericycle, conjuctive tissue, xylem, phloem and pith parenchyma. 200 The juveniles oiMeloidogyne incognita penetrated at or behind the root tips of Momordica charantia. They migrated intercellularly, in the inner tissue, by separating the cell walls. The giant cells were induced in the region of undifferentiated phloem within 48h of inoculation. Maximum number of nuclei and highly dense cytoplasm was noticed after 12 days of inoculation. Decrease in number of nuclei and increase in vacuolation was found after 18 days of inoculation. Smaller giant cells became empty and changed into vessel like elements by the deposition of lignified secondary wall material. Larger giant cells with little or no cytoplasm also transformed into abnormal vessel elements, after 30 days of inoculation. Secondary infection was noticed after 30 days of inoculation. The egg masses of all the females were not expelled out of the plant tissue. Some egg masses remained inside. Eggs of these egg masses hatched and the second-stage juveniles, traversing intercellularly through cortical cells, reached cambial zone and caused secondary infection. Repetition of hypertrophic and hyperplastic reactions, due to secondary infection, caused a rapid increase in gall size. Some freshly hatched second-stage juveniles were found associated with preformed gaint cells. These juveniles, instead of inducing new giant cells, started feeding on old giant cells. Hypertrophic and hyperplastic reactions were accompanied with such type of feeding. As far as abnormalities in vascular elements are concerned, the abnormality in orientation of vascular strands was started 48h after 201 inoculation.Vascular strands were severely distorted due to the multiple hypertrophic and hyperplastic reactions taking place continuously. The strands became wavy and appeared as scattered patches,when seen in longitudinal sections. The vessel elements became structurally abnormal within 48h of inoculation. The vessel elements broadened near giant cells due to hypertrophic reactions. Irregular shape and size of vessel elements of metaxylem strands are the result of pressure exerted on them by the tissue resulting from hypertrophic and hyperplastic reactions. Abnormal origin of vessel elements was observed 72h after inoculation from small parenchyma cells; 12 days after inoculation from hypertrophied parenchyma cells; 18 days after inoculation from small giant cells; and 30 days after inoculation from larger giant cells. From these observations, it is suggested that formation of abnormal vessel elements in excess amount might increase upward translocation, or supply water to giant cells, or provide protection to giant cells, or gives support to entire gall to prevent its collapse. 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