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University Mk:rorilms International 300 N. Zeeb Road Ann Arbor, Ml 48106

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Alves de Lima, Marinez Muraro

PHYSICAL AND CHEMICAL FACTORS AFFECTING REPRODUCTION OF PRATYLENCHUS SPECIES IN MONOXENIC CULTURE

The Ohio State University Ph.D. 1982

University Microfilms International300 N. Zeeb Road, Ann Arbor, MI 48106

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University Microfilms International

PHYSICAL AND CHEMICAL FACTORS AFFECTING REPRODUCTION OF PRATYLENCHUS SPECIES IN MONOXENIC CULTURE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

by Marinez Muraro Alves de Lima, B.S., M.S.

*****

The Ohio State University 1982

Reading Committee: Approved By Dr. R. M. Riedel Dr. L. Rhodes Advisor Dr. I, Deep Department of Plant Pathology This dissertation is dedicated to my parents, Isaltina and Roberto, and to my husband, Abdalla, for their constant concern, encouragement and support. ACKNOWLEDGEMENTS

I thank my colleagues, the graduate students, staff and faculty members of the Department of Plant Pathology for providing such a warm and friendly environment conducive to learning. I extend my sincere thanks to Dr. R. M. Riedel for his advice and criticism during the conduction of the research and review of the manuscript; to Dr. M. J. Martin for her suggestions, supply of nematode stock cultures and incentive during the research; and to R. D. Thames for her generous help and encouragement. My sincere appreciation is reserved to the Instituto Agronomico de Campinas, Bnpresa Brasileira de Pesquisa Agropecuaria - EMBRAPA and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq, for granting me this invaluable opportunity to further my education. VITA

November 16, 1944 Born - Sao Paulo-SP, Brazil 1967 B.S., Escola Superior de Agricultura "Luiz de Queiroz," Piracicaba-SP, Brazil 1980 M.S. The Ohio State University, Columbus, Ohio

FIELD OF STUDY Major Field: Plant Pathology Research Interest: Plant Tissue Culture Applied to Plant Pathology TABLE OF CONTENTS

Page DEDICATION...... ii ACKNOWLEDGEMENTS...... iii VITA ...... iv LIST OF...... TABLES ...... vi LIST OF FIGURES...... viii LIST OF PLATES...... ix INTRODUCTION ...... 1 Chapter I. PHYSICAL AND CHEMICAL FACTORS AFFECTING REPRODUCTION OF PRATYLENCHUS PENETRANS IN MONOXENIC CULTURE...... 3 Introduction and Literature Review...... 3 Materials and Methods...... 9 Results...... 16 Discussion...... 37 II. COMPARATIVE REPRODUCTION OF FOUR SPECIES OF PRATYLENCHUS ON ALFALFA CULTURE...... 47 Introduction and Literature Review...... 47 Materials and Methods ...... 49 Results...... 52 Discussion...... 60 BIBLIOGRAPHY...... 65 APPENDIX -- Composition of Riedel's et al. (1973) and Murashige and Skoog (196‘ZJ’ me d i a ...... 71

v LIST OF TABLES

Reproduction of Pratylenchus penetrans on four plant tissues cultured on three different media...... 20 Reproduction of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et alY medium at three yeast extract concentrations ...... 21 Reproduction of Pratylenchus penetrans on alfalfa seedlings cultured on Riedelfs et al. medium at three yeast extract concentrations...... 22 Reproduction and composition of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium at three yeast extract concentrations...... 23 Reproduction of Pratylenchus penetrans on pea roots cv. 'Extra Early Alaska' cultured on Riedel's et al. medium at three yeast extract concentrations...... 24 Reproduction of Pratylenchus penetrans on pea roots on pea roots of two cultivars cultured on Riedel’s et aL. medium...... 25 Populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium in three different kinds of containers...... 26 Populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel*s et al. medium in different kinds of containers...... 27 Population density of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium in different kinds of containers ...... 28 Reproduction of four Pratylenchus species on alfalfa callus cultured on Riedel's et al. medium at 25 C for 100 days ...... 54 Reproduction of four Pratylenchus species on alfalfa on alfalfa callus cultured on Riedel’s et al. medium at 25 C for 120 days...... 55

vi Table Page 3.1. Composition of Riedel*s et al. medium (1973)...... 72 3.2. Composition of Murashige and Skoog's medium (1962)...... 73

vii LIST OF FIGURES Figure Page 1.1. Reproduction of Pratylenchus penetrans on Riedel's et al. medium with 1 g yeast extract/1 at 25 C. A) for a 80-day period; B) for a 110-day period...... 29 1.2. Reproduction of Pratylenchus penetrans on Riedel's et al. medium with 0.5 g yeast extract/1 at 25 C. Aj “For a 80-day period; B) for a 110-day p e r i o d...... 30 1.3. Reproduction of Pratylenchus penetrans on Riedel's et al. medium without yeastextract at 25 C . A7 Tor a 80-day period; B) for a 110-day period...... 31 2.1. Reproduction of Pratylenchus brachyurus on Riedel's et al. medium at“25 C. A) Tor a 100-day period; For a 120-day p e r i o d ...... 56 2.2. Reproduction of Pratylenchus crenatus on Riedel's et al. medium at 25 C. A) for a 100-day period; B] Tor a 120-day p e r i o d ...... 57 2.3. Reproduction of Pratylenchus penetrans on Riedel's et al. medium at 25 C. A] for a- lOO’-day period; B) for a 120-day period ...... 58 2.4. Reproduction of Pratylenchus scribneri on Riedel's et al. medium at'""25 C. A) for a 100-day period; B) Tor a 12-day pe r i o d ...... 59

viii LIST OF PLATES Plate Page I. Inoculation of aseptic nematode suspension into French square bottles in a laminar airflow cabinet...... 13 II. Baermann funnels made from styrofoam cups and cheese-cloth used for nematode extraction...... 14 III. Longitudinal section of alfalfa rot infected by Pratylenchus penetrans with damaged area and nematodes around vascular region ...... 32 IV. Pratylenchus penetrans in alfalfa root with labial region penetrating cell wall...... 33 V. Intra- and intercellular penetration of alfalfa roots by Pratylenchus penetrans...... 34 VI. Transverse section of masses of Pratylenchus penetrans in parenchymatous-like callus-cells of alfalfa r o o t s ...... 35 VII. Pratylenchus penetrans feeding on xylem cells of alfalfa r o o t s ...... 36

ix INTRODUCTION

Byars (3) in 19X4 was the first to establish a monoxenic culture of nematodes by sterilizing root-knot nematode eggs and inoculating them onto tomato and cowpea roots. The nematodes were maintained asep- tically through one generation in the seedling cultures. In 1933, Tyler (62) also cultured root-knot nematodes on tomato seedlings to study reproduction without males. Mountain (34, 35) cultured Pratylen­ chus minyus on sterile, excised c o m roots, representing the next sig­ nificant advance in culturing plant parasitic nematodes. But it was Krusberg’s work (25) that provided a break-through for those inter­ ested in some of the more important migratory parasitic nematodes, and ample use has been made of the technique in a wide range of physiolo­ gical, taxonomic and pathogenicity studies (44). However, monoxenic cultures have still wider applications, and their use could be expanded if the techniques used to maintain those cultures were not so time- consuming. In fact, in a survey of the use of monoxenic cultures among nematologists (26), there was a common sense that if more simplified and less time-consuming techniques were available, a more intensive use of those cultures in research would be possible. There was also a general claim that if the subcultures necessary for the maintenance of the cultures were less frequent, it would be another incentive to the use of monoxenic cultures.

1 The first chapter of this work deals essentially with monoxenic culture of Pratylenchus penetrans and the physical and chemical factors affecting reproduction. The objective was to study some of the steps involved in the establishment and maintenance of the cultures. These basically included the search for a better plant host-medium combina­ tion than the one in most common use, and the influence of containers on short and long term maintenance of cultures. Histology of alfalfa tissue cultured on callus-inducing medium complemented the study, giv­ ing an indication of nonatode distribution in callused tissue. Work reported in the second chapter compares reproduction of four Pratylenchus species, F\ brachyurus, P. crenatus, £. penetrans and P. scribneri on alfalfa callus culture. This work was undertaken in the hope that understanding the basic influence of some cultural techniques on nematode reproduction would lead to the development of more efficient techniques for culturing Pratylenchus species monoxenically. CHAPTER I

Physical and chemical factors affecting reproduction of Pratylenchus penetrans in monoxenic culture

INTRODUCTION AND LITERATURE REVIEW A list of 41 species of plant parasitic nematodes cultured on plant tissues was included in a recent review (26). Six additional species of Pratylenchus (6, 31, 32, 47) and two more species in other genera (52, 62) have been cultured on monoxenic culture since. Although Pratylenchus is the genus from which the largest number of species has been cultured, in-depth studies on culturing techniques have been conducted mostly for Ditylenchus dipsaci (4, 15, 17, 25, 64). Viglierchio et al. (64), for instance, studied axenization of several host plants, various culture media, and reproduction of D. dipsaci on tissues of different age and type of several hosts. Eriksson (15) and Bingefors and Bingefors (4) detailed equipment and procedures for cul­ ture and maintenance of different D. dipsaci races. Faulkner et al. (17) described techniques for mass rearing of D. dipsaci to be used in a plant breeding program. Krusberg (25) studied histopathology of alfalfa cultures infected with D. dipsaci. In the case of Pratylenchus penetrans, although many aspects have been studied, data are found more scattered in the literature. Schroeder and Jenkins (50) studied reproduction of P. penetrans on

3 4 excised roots and callus from 11 crop plants on three culture media. Nematode reproduction was highest on callus tissue increasing from 9 to 345-fold, while the maximum increase on excised roots was 46-fold. Pea, cucumber and alfalfa were the callus tissues presenting higher repro­ duction. Schroeder (49) studied reproduction of P. penetrans on corn roots cultured on two media, and reported higher reproduction rate on Tiner’s medium, when compared to White’s. Krusberg and Blickenstaff (27) studied the influence of auxins and kinetin on reproductive rates and found that £. penetrans yielded the largest number of nonatodes on media containing 2,4-dichlorophenoxy- acetic acid (2,4-D) and 2,4-D + naphthalene acetic acid (NAA), whereas reproduction on medium with kinetin was reduced. Riedel and Foster (41) cultured D. dipsaci and P. penetrans on alfalfa callus growing on modified Krusberg and White media. Highest populations on £. penetrans were found on Krusberg and White media with 2,4-D and <*-NAA. The authors suggested that White medium was a con­ venient alternative for production of £. penetrans. Riedel et al. (43) studied the effect of varying each component in a four-component basic medium composed of yeast extract, 2,4-D, sucrose and agar. In 1973 the same authors (43) reported a simplified medium, based on the studies mentioned above, to culture P. penetrans and D. dipsaci. Mitsui et al. (32) studied temperature and pH effect on the propa­ gation of 22 populations of seven species of Pratylenchus cultured on alfalfa callus tissue. The effect of three different media on the 5 propagation of seven Pratylenchus species in culture was also studied by Mitsui (31). Monoxenic cultures of nematodes can be regarded as an approach to pure culture useful in obtaining high populations of sterile nematodes. Tissue cultured nematodes have been used to study various aspects of their biology, physiology and ultrastructure (44). Two basic areas of phytopathological research, establishment of pathogenicity and crop loss assessment, are particularly difficult to pursue when large popu­ lations of microbiologically sterile nematodes are not available. For the nematologist these cultures are considered as a tool for his work. His ultimate interest is to maintain the specimens in col­ lection and/or propagate them whenever high populations are necessary for specific studies. The simplest routine techniques are desirable. Factors which affect the ease of culture maintenance include host tissues, media on which host callus are produced and containers in which cultures are maintained. Besides good culturing qualities which favor high nematode repro­ duction and/or long term maintenance of cultures, the host plant tissue should be easily available, as well as easy to sterilize and to handle. A simplified medium or a formulation which can be acquired ready-made are highly desirable for the routine maintenance of nematode cultures. Containers to be used should be easily handled, space effi­ cient, and most of all, adequate for the objective of the culture; that is, either long term maintenance or high population production. The site of nanatode feeding and reproduction in monoxenic cul­ tures of Pratylenchus spp. is an unanswered question. Cultures are 6 usually established from seedlings or excised roots cultured on medium with a callus inducing substance, usually 2,4-D. In this case not pure callus, but a mixed tissue composed of callus cells and organized tis­ sue is obtained, and, among nematologists, referred to as "callus tissue." Pratylenchus species reported to have been cultured have been propagated in those kinds of culture without any follow-up to determine in which type or types of tissue the nematodes actually feed. Other plant parasitic nematodes, including Ditylenchus dipsaci, D. destructor and Aphelenchoides ritzemabosi are known to feed and reproduce on parenchyma-like callus cells. Thus, D. dipsaci was found scattered through the loose parenchyma callus cells of alfalfa callus (25). This species seems to multiply on callus independently of its origin making a non-host or resistant plant species or variety lose its resistance (4, 14, 15). On the other hand, populations apparently differ with the ease they establish in callus tissue, sometimes making necessary the use of sterile seedlings (14). Viglierchio et al. (64) found some stem nematode biotypes to reproduce far better on organized tissue than on parenchyma callus cells. D. destructor was reported to reproduce well on cultures of callus tissues of potato, carrot, clover and tobacco (9, 16). Krusberg (25) reported A. ritzemabosi to feed and reproduce on the loose, parenchyma cells of alfalfa callus. The obvious advantage in determining in which types of tissue nematodes feed is to be able to manipulate the cultures in such a way as to favor production of the type most adequate for nematode feeding and reproduction. 7 Data in the literature establishing correlations between increase in tissue growth and nematode population are equivocal. For instance, Aphelenchoides ritzemabosi reproduction has been reported positively (66) and negatively (65) correlated to increase in callus growth. Krusberg and Blickenstaff (27), studying influence of plant growth substances on Ditylenchus dipsaci, Pratylenchus penetrans and P. zeae cultured on alfalfa callus, concluded that the medium supporting best callus growth did not necessarily support best nematode reproduction. In the case of Pratylenchus, Schroeder and Jenkins (50) studying repro­ duction of £. penetrans on 11 crop plants found no correlation between nematode reproduction and amount of tissue growth. Likewise Mitsui (31), comparing reproduction of seven Pratylenchus species, including P. penetrans, on alfalfa callus produced on different media, did not find a correlation between reproduction and tissue growth. The broad objective of this study was to develop more simplified, efficient and convenient techniques to obtain maximum P. penetrans populations, and also to determine the effect of containers on main­ tenance of cultures. With such a purpose, the following points were examined: a) comparative reproduction of P. penetrans on four different plant substrates, potato, carrot, pea and alfalfa, cultured on three different media; b) reproduction of P. penetrans on Riedel et al. (42) medium with varying concentrations of yeast extract; c) comparative reproduction of £. penetrans on different pea cultivars; 8

d) effect of different containers on short and long t e m storage of P . penetrans, and e) investigation on feeding and reproduction site of P. penetrans on alfalfa cultures. MATERIALS AND METHODS

A. Experimental Plan

The same experimental design was used throughout the study; only the number of treatments varied. The basic replicate in the design was a single container, with two pieces of pea roots, or three pieces of potato tuber or carrot root, or three alfalfa seedlings, depending upon the plant species studied. Each treatment consisted of 10 repli­ cates, unless otherwise specified. Containers were arranged in a com­ pletely randomized design. The experiments were done twice.

B. Inoculum Source

Pratylenchus penetrans used in this study was obtained originally from Prof. V/. F. Mai, Plant Pathology Department, Cornell University, Ithaca, NY. These cultures originated from a single female isolated from apple roots collected in New York State. Stock cultures were maintained at 20 C on alfalfa callus using methods of Riedel and Foster (41], and subcultured every three months.

C. Sterilization of Plant Tissues and Bioassay for Contamination

Alfalfa (Medicago sativa L. ’Ranger’) seeds were immersed in con­ centrated sulfuric acid (36 N) for 15 min, rinsed three times in sterile, double distilled water, immersed in 1:1000 mercuric chloride

9 in 30% ethanol for 15 min, and rinsed three times in sterile, double distilled water. To bioassay for contamination and for germination, seeds were incubated on potato dextrose agar (Difco Laboratories, Detroit, MI 48232) for seven days under natural day-light conditions. Three seedlings were used for each container. Potato (Solanum tuberosum L. 'Norchip') tubers and carrot (Daucus carota L. 'Danvers 126') roots were washed with a weak Alconox solu­ tion and scrubbed clean. Potato tubers were immersed for 10 min, in 1% sodium hypochlorite solution, and carrot roots, for 20 min. Cylin­ ders of 6 mm diameter were obtained by inserting a sterile cork borer longitudinally along the cambial region in each tuber or root. After removing the ends, these cylinders were washed in sterile, double dis­ tilled water, and cut into ca. 2 mm thick discs. These discs were incubated on potato dextrose agar medium for seven days, to bioassay for contamination. Three discs of each species were used for each con­ tainer . Pea (Pisum sativum L . ' Little Marvel * and ' Extra Early Alaska') p seeds were washed with a weak Alconox solution, treated with 5.25% sodium hypochlorite solution for 10 min, rinsed three times with sterile, double distilled water. Then seeds were incubated for seven days on potato dextrose agar medium to bioassay for contamination and germination. Roots were excised below the hypocotyl region, and two of each placed in each container filled with callusing medium. 11 D. Containers and Callus ing Medium

Basically, four types of containers were used: i) 25 X 150 mm glass tubes closed with plastic caps or PVC films; ii) 30 ml French square bottles closed with plastic caps or PVC films; iii) 10 cm dia- meter plastic petri dishes sealed with Parafilm (American Can Co., Greenwich, CT 06830); and iv) 250 ml jars with metal lids or sealed with PVC films. Ibbes (slanted) and French square bottles were filled with 15 ml of medium, and plastic petri dishes and jars with 30 ml of medium. Tubes were placed inside plastic bags to facilitate storage. Only French square bottles were used throughout the studies, except for the experiments specially designed to study containers. The media used were 1% water agar (WA), Riedel et al. medium (P.) (42) (Appendix, Table 3.1), and Murashige and Skoog medium (MS) (36) (Appendix, Table 3.2) with the macromineral concentrations reduced to one-half strength. Concentration of 2,4-D used for carrot, pea, alfalfa and potato cultures on MS medium were 0.5, 0.5 , 2 and 5 mg/1, respectively. Nematode reproduction on alfalfa seedlings cultured on R medium with varying yeast extract concentrations (0.0, 0.5 and 1 g/1) was tested.

E. Extraction of Nematodes from the Stock Cultures

To obtain aseptic inoculum, Baeimann funnels made from 23 cm diameter pie pans were autoclaved inside paper bags at 1 atm 12 (121 C) for 20 min. Nematodes were extracted aseptically for 48 hr from stock cultures in a laminar airflow cabinet.

F. Inoculation

Aseptic nematode suspension extracted from stock cultures was dis-

T> pensed into containers with a Finnpipette (Ky Finnpipette, Helsinki), in a laminar airflow cabinet (Plate I). The volume of the suspension delivered was fixed according to the nematode level desired. Volume ranged from 0.2 ml to 0.5 ml per container. At least 10 counts of the representative aliquots were made to estimate the initial inoculum.

G. Incubation Conditions

The containers were incubated at 25 C in the dark, except for the experiment with different types of tissue which were incubated at room temperature (20-30 C).

H. Extraction, Counting and Data Analysis

Nematodes from each container were extracted for 24 hr using Baermann funnels made from 250 ml styrofoam cups and cheese-cloth (Plate II). Tissue and agar medium from each container were extracted separately. TWo, 5 ml aliquots of the nematode suspension extracted from tissue and agar were counted and averaged to determine the total nanatode number in each container. Extractions were processed at different periods of time for each experiment. Thus, P. penetrans reproduction was evaluated as follows: Plate I. Inoculation of aseptic nematode suspension into French square bottles in a laminar airflow cabinet. Plate II. Baermann funnels made from styrofoam cups and cheese-cloth used for nematode extraction. 15 1) four host tissues cultured on three different media, 50 days after inoculation; 2) alfalfa seedlings cultured on R medium at three yeast extract levels, 20, 40, 60 and 80 days (first experiment), and 20, 50, 80 and 110 days (second experiment), after inoculation; 3) pea roots cultured on R medium at three yeast extract levels, 20, 40, 60 and 80 days after inoculation; 4) comparison of two root cultivars, 30, 60, 90 and 120 days after inoculation; 5) short term maintenance of cultures in different containers, 70 and 140 days after inoculation; 6) long term maintenance of cultures in different containers, 5 and 10 months after inoculation. Data obtained were transformed to log x or log x+1, prior to ana­ lysis of variance. Treatment mean comparisons were done using Duncan's multiple range test. Regression analyses were also used, when appli­ cable.

I. Preparation of Plant Tissue for Histological Examination

For histological study, 6 month-old stock cultures of Pratylenchus penetrans maintained at 20 C on alfalfa callus and cultured by method of Riedel and Foster (43) were used. Alfalfa roots were fixed in FAA, then cut into 5 mm sections, dehydrated in tertiaryl butyl alcohol (22), and embedded in paraffin. Roots were sectioned at a thickness of 12 mm with a rotary microtome. Sections were mounted on glass slides and stained with safranin and fast green (22). RESULTS

Fifty days after inoculation, reproduction of Pratylenchus pene­ trans was highest on R medium for all four types of tissue used (Table 1.1). Nematode populations Tanged from 6841 to 1080 on R medium, from 1211 to 510 on MS, and from 123 to 539 on WA medium. Alfalfa seedlings cultured on R medium proved to be the best substrate, followed by pea and carrot roots cultured on the same medium. Nematode reproduction and tissue fresh weight were the poorest on WA medium. Most fresh weight of host tissue was obtained on MS medium in all cases. A nega­ tive correlation was found between callus fresh weight and nematode number (Table 1.1). Reproduction of P. penetrans on alfalfa seedlings cultured on R medium at three different concentrations of yeast extract (Table 1.2) proved to be significantly higher on medium with 0.5 and 1 g yeast extract/1 during the 80-day period studied. However, for the 110-day period (Table 1.3), mean nematode population on medium with 0.5 g yeast extract/1 at 110 days was 317, a value significantly lower than the one found for 1 g/1 yeast extract at the same extraction. Results of the regression analysis performed for both experiments (Tables 1.2 and 1.3) relating nematode production with time, for each concentration of yeast extract and for both period studied, are repre­ sented in Figures 1.1, 1.2 and 1.3.

16 17 On R medium containing 1 g yeast extract/1, for the 80-day period, reproduction related to extraction time fit a linear regression des­ cribed by the equation Y = 1.459 + 0.02437X, with r2 = 0.9949 (Figure 1.1-A), whereas for the 110-day period, a quadratic model was found to fit the reproduction-time relationship, represented by the equation Y = 0.675 + 0.05046X - 0.000295X2, with r2 = 0.9993 (Figure 1.1-B). Estimated maximum nematode population occurred at 86 days in the 110- day cultures. Reproduction of £. penetrans related to time on R medium with 0.5 g yeast extract/1 for the 80-day period and 110-day period are represented by the equations Y = 1.5075 + 0.01963X, with r2 = 0.9860 (Figure 1.2-A) and Y = 0.756 + 0.0494X - 0.00031X2 with r2 = 0.9981 (Figure 1.2-B), respectively. Estimated maximum nematode population for the 110-day period occurred at 80 days. On R medium without yeast extract, nematode reproduction related to the periods of 80 and 110 days aTe described by the equations Y = 1.167 + 0.015775X, with r2 = 0.9415 (Figure 1.3-A), and Y = 1.061 + 0.0268X - 0.0002X2, with r2 = 0.9971 (Figure 1.3-B). Estimated maximum population during the 110-day period occurred at 67 days. Analysis of the population structure of P. penetrans on alfalfa seedlings cultured on R medium at three yeast extract levels for 80 days is given on Table 1.4. During this period female-male ratios were close to 1. Reproduction of P. penetrans on ’Extra Early Alaska’ pea roots (Table 1.5) was so erratic that coefficients of variation reached 18 values around 25%. Forty days after inoculation the mean population size had not reached the number of nematodes used as initial inoculum. Comparison of P. penetrans reproduction on two pea cultivars showed a 10 fold higher population on 'Little Marvel' when compared to 'Extra Early Alaska' (Table 1.6), indicating that the nematodes devel­ oped differently in the pea cultivars. When P_. penetrans reproduction on three different kinds of con­ tainers, 25 X 150 mm tubes, 30 ml French square bottles and 10 cm dia­ meter plastic petri dishes, was compared, significantly higher popula­ tions were obtained on bottles, averaging 594 and 542 nematodes (Table 1.7), for 70 and 140 days after inoculation, respectively. Reproduc­ tion of cultures contained in tubes decreased nearly 35% from 70 to 140 days after inoculation. In cases of longer teim storage (Table 1.8), comparing 25 X 150 mm tubes closed with plastic caps or PVC films, 30 ml French square bot­ tles closed with plastic caps or PVC films, 10 cm diameter plastic petri dishes and 250 ml jars, cultures contained in petri dishes showed significantly higher reproduction at the end of 5 and 10-month storage (Table 1.8). Five months after inoculation populations in petri dishes were 2 to 3 times higher when compared to bottles and tubes, and almost 6 times, when compared to jars. In 10-month storage cultures in petri dishes yielded 1728 nematodes against about 500 in bottles and 150 in tubes, whereas cultures in jars averaged 28 nematodes. A great deal of desiccation characterized the treatments in petri dishes and jars, mostly at 10-month storage. This was responsible for the complete loss 19 of nematodes in 50% of the replicates in petri dishes (Table 1.8). Medium in jars was reduced to 1/3 volume and tissues were apparently completely dried out. The use of PVC films as a substitute for plastic caps for tubes and bottles did not result in any increase in nematode populations (Table 1.8). When data on nematode populations contained in French square bot­ tles and tubes with plastic caps, and petri dishes (Table 1.8) were 2 transformed to nematode number per cm of container surface (Table 1.9), petri dishes gave the lowest population density, with 37 and 39 nema- 2 todes per cm , 5 and 10 months after inoculation, respectively. Examination of paraffin sections of alfalfa roots showed that P. penetrans was found in parenchyma cells of root cortex (Plates III, IV and V), parenchyma-like callus cells (Plate ATE), and occasionally in vascular tissue (Plate VII). In the sections examined, about 30% of the nematodes were found in parenchyma cells, and 70% around vascular tissue. All nematode stages, including eggs, were found. Cell walls at feeding sites were thicker and stained darker with safranin (Plate III). Nematode location within the tissue was inter- and intracellular (Plate IV). There was much cell wall breakage. Cell walls were fre­ quently absent from the area occupied by the nematode (Plate V ) . Nematodes in callus cells occurred sometimes in masses (Plate VI), sometimes scattered among callus cells. Occasionally nematodes were found feeding next to xylem cells (Plate VII). Table 1.1. Reproduction of Pratylenchus penetrans on four plant tissues cultured on three different media.

Alfalfa 'Ranger1 Carrot 'Danvers 126' Pea 'Little Marvel' Potato 'Norchip'

WAP R MS m R MS WA R MS WA R MS

Callus Fresh Weight 6lSc 283y 559z 13 9x 278y 533z 87x 228y 382z 191x 323x 833y (MS)

Nematodes/ 10 cultures 170ri 6841a 514h 454g 2865b 51 Of 123j 2790b 1211c 539f 1080d 691e

p WA = \% water agar medium; R = Riedel's medium; MS = Murashige and Skoog modified medium, q The values followed by the same letter are not significantly different according to Duncan's multiple range test at P = 0.01. r The values followed by the same letter are not significantly different according to Duncan's multiple range test at P = 0.01. Data were transformed to log x or log x+1 prior to analysis. 21

Table 1.2. Reproduction of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium at three yeast extract concentrations for 80~"3ays.

nematodes/10 cultures^ Tv,Ve aftpr grams yeast extract/1______inoculation• ■Y r1 r. oTo 075 570

20 56 bz 138 a 130 a 40 74 b 220 a 354 a 60 153 b 663 a 1097 a 80 490 b 1339 a 2816 a

Each culture consisted of callus from three alfalfa seedlings; the initial inoculum was 158 nematodes which is the mean number of nematodes in 10 aliquots of inoculum. Values in a horizontal row followed by the same letter are not sig­ nificantly different according to Duncan's multiple range test at P = 0.01. Data were transformed to log x or lox x+1 prior to analysis. 22

Table 1.3. Reproduction of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel’s et al. medium at three yeast extract concentrations for 110 days.

Nematodes/10 cultures^ no,,,. grams yeast extract/1 uays arter -n— ?j------p------t— tt inoculation u*u u*b ,u

20 38 a2 46 a 48 a 50 122 b 296 a 317 a 80 198 b 617 a 844 a 110 83 c 317 b 506 a Each culture consisted of callus from three alfalfa seedlings; the initial inoculum was 67 nematodes which is the mean number of nema­ todes in 10 aliquots of inoculum. Values in a horizontal row followed by the same letter are not sig­ nificantly different according to Duncan’s multiple range test at P = 0.01. Data were transformed to log x or log x+1 prior to anal- ysys. 23

Table 1.4. Reproduction and composition of populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel1s medium et al. at three yeast extract concentrations.

grams of yeast extract/1 Days after 0.0 0.5 1.0 inoculation F M L F ML F M L

20 17.7Z 15,9 66.4 15.4 15.8 68.8 24.1 17.6 58.2 40 27.5 21.5 51.0 28.6 26.6 44.8 29.8 24.2 46.0 60 24.8 25.5 49.7 19.1 21.1 59.8 23.8 23.1 53.1 80 18.1 21.5 60.3 17.6 17.8 64.5 15.9 15.6 68.4 y F = female, M = male , L = larvae z Percent values of each stage over the total population. 24

Table 1.5. Reproduction of Pratylenchus penetrans on pea roots cv. 'Extra Early Alaska1 cultured on Riedel et al. medium at tliree yeast extract concentrations.

Nematodes/10 cultures^ grams yeast extract/1 Days atter 0 5 ------075------770" inoculation

20 4 a 8 a 14 a 40 10 a 45 a 53 a 60 10 c 187 a 548 a 80 28 c 385 b 1014 a

Values are the mean number of nematodes in 10 cultures, where each culture consisted of too pea roots; the initial inoculum was 158 nematodes which is the mean number of nematodes in 10 aliquots of inoculum. Values in a horizontal row followed by the same letter are not dif­ ferent according to Duncan's multiple range test at P = 0.01. Data were transformed to log x+1 prior to analysis. Table 1.6. Reproduction of Pratylenchus penetrans on pea roots of too cultivars cultured on Riedel's et al. medium.

Days after inoculation ’Little Marvel' 'Extra Early Alaska'

30 44 az 39 b 60 94 a 2 b 90 136 a 12 b 120 630 a 61 b y Each culture consisted of callus from too pea roots; the initial inoculum was 46 nanatodes which is the mean number of nonatodes in 10 aliquots of inoculum, z Values in a horizontal row followed bythe same letter are not dif­ ferent according to Duncan’s multiple range test at P = 0.01. Data were transformed to log x or log x+1 prior to analysis. 26

Table 1.7. Populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium in three different kinds of containers.

Nematodes/10 cultures Days after Tubes Petri inoculation Bottles Dishes

70 422ycZ 594 a 424 b 140 276 b 542 a 495 a

Container specifications: T = 25 x 150 mm tubes with plastic caps, B = 30 ml French square bottles, PD = 10 cm diameter plastic Petri dishes. Each culture consisted of callus from three alfalfa seedlings; the initial inoculum was 22 nematodes which is the mean number of nematodes in 10 aliquots of inoculum. Values in a horizontal row followed by the same letter are not significantly different according to Duncan's multiple range test at P = 0.01. Data were transformed to log x or log x+1 to analysis. Table 1.8. Populations of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel's et al. medium in different kinds of containers.

Months after inoculation Nematodes/10 cultures Tubes Bottles Petri Jars PVC caps PVC caps Dishes

5 501wbx 485 b 550 b 535 b 1605 a 280 c

10 177 c 108 c 540 b 482 b 1728ya 28zd v Container specifications: 25 x 150 mm tubes sealed with plastic caps or PVC films; 30 ml French square bottles sealed withplastic caps or PVC films; 10 cm diameter plastic petri dishes sealed with Parafilm; 250 ml jars closed with metal lids plus PVC film, w Values are the mean number of nematodes in 10 cultures, unless otherwise indicated, where each culture consisted of callus from three alfalfa seedlings; the initial inoculum was 34 nematodes, which is the mean number of nematodes in 10 aliquots of inoculum, x Values in a horizontal row followed by the same letter are not significantly different according to Duncan’s multiple range test at P - 0.01. Data were transfoimed to log x prior to analysis, y Value is the mean number of nematodes in five cultures, z Value is the mean number of nematodes in nine cultures. 28

Table 1.9. Population density of Pratylenchus penetrans on alfalfa seedlings cultured on Riedel’s et al.medium in different kinds of containers.

Months after Containers" inoculation Tubes Bottles Petri Dishes

5 86xby 99a 37 c 10 22 b 68a 39zc w Container specifications: 25 X 150 mm tubes with plastic caps; 30 ml French square bottles with plastic caps; 10 cm diameter plastic petri dishes sealed with Parafilm. 2 x Values are the mean number of nematodes per cm in 10 cultures, unless otherwise indicated, where each culture consisted of callus from three alfalfa seedlings; the initial inoculum was 34 nematodes, which is the mean number of nematodes in 10 aliquots of inoculum, y Values in a horizontal row followed by the same letter are not sig­ nificantly different according to Duncan’s multiple range test at P = 0.01. Values were transformed to log x prior to analysis, z Value is the mean number of nematodes in five cultures. 120 29 100 80 60 days of incubation r2 •= 0.9993 •= r2 Y = 0.675 + 0.05046X 0.000295X- r2 = 0.9949 Y = 1.459 + 0.02437X 20 * Each* point mean the is of replications.10 period; period; B) for a 110-dayperiod. mediumat gyeast 1 extract/1 at 25 C. A) for a 80-day 0 10 10 100 10G 1,000 1,000 10,000 10,000 Figure 1,1. Figure 1,1. Reproductionof Pratylenchus penetrans Riedel'son et al. nematodes/container nematodes/container 120 30 100 80 60 days of incubation 1+0 r2 = 0.9981 r2 = r2 0.9860 Y = 0.756 0.0494X+ - 0.00031X Y = 1.5075 + 0.01963X 20 ♦ Each periodEach meanis the of ♦ 10 replications. 80-day period; 80-dayperiod; B) for a 110-dayperiod. mediumwith yeast 0.5 g extract/1 at C 25 . A) for a . 0

10 10 100 100 1,000 1.00C 10,000 10,000

nematod.es/container nematodes/container Figure 1.2. Reproduction of Pratylenchus penetrans onRiedel's et al. 120 31 100 80 60 days of incubation Y = 1.061 0.02684+ 0.0002X- r2 0.9415 = Y = 1.167 0.015775X+ 20 * Eachpoint mean the is * of 10 replications. period; period; B) for a 110-dayperiod. mediumwithout yeast extract at C. 25 A) for a 80'rHay 0 . 10 10 100 100 1,000 1,000 10,000 10,000

nematodes/container nematodes/container Figure 1.3. Reproduction ofPratylenchus penetrans onRiedel's et al. Plate III. Longitudinal section of alfalfa root infected by Pratylenchus penetrans with damaged area and nematodes around vascular region. Plate IV. Pratylenchus penetrans in alfalfa root with labial region penetrating cell wall. Plate V. Intra and intercelluluar penetration of alfalfa roots by Praty­ lenchus penetrans. Plate VI. Transverse section of masses of Pratylenchus penetrans in parenchymatous callus cells of alfalfa roots. Plate VII. Pratylenchus penetrans feeding on xylan cells of alfalfa roots. DISCUSSION

Desirable characteristics for host tissue used as substrate for culturing nematodes include easy handling, good support of nematode population and good maintenance of populations with a minimum require­ ment for subcultures. Among the treatments studied (Table 1.1], alfalfa seedlings cultured on R medium proved to be the best substrate-medium combination. However, although alfalfa is the usual substrate, for monoxenic cultures, each time a subculture is needed, alfalfa seeds have to go through the whole protocol of surface sterilization, bioassay for contamination, transfer to callus induction medium and inoculation, a time consuming procedure. Pea and carrot roots on R medium were the second best substrate for P. penetrans. Schroeder and Jenkins (50) in an earlier study of P. penetrans reproduction on excised roots and callus tissue of 11 crop plants cultured on three media also found higher nematode populations on alfalfa and pea cultured on Krusberg’s medium, although no statisti­ cal analysis of those results was presented. As pea seeds are easily handled, an attempt was made to confirm the suitability of pea roots as an alternative to alfalfa, a possibility suggested by the results obtained in Table 1,1. However, instead of 'Little Marvel', which pre­ sented promising results before (Table 1.1), another pea cultivar, 'Extra Early Alaska', was used, and on this cultivar P. penetrans popu­ lations were very low and variable (Table 1.5). It was then postulated

37 38 that different pea cultivars might present different degrees of suscep­ tibility to the nematodes, although in many instances plant tissues were reported, to lose specificity when cultured on callus inducing medium (4, 25, 27, 64, 66). Comparing P. penetrans reproduction on 'Little Marvel* and 'Extra Early Alaska* (Table 1.6) it became apparent that indeed the two pea cultivars presented different degrees of susceptibility to the nematode. The fact that the four kinds of host tissue placed on WA medium provided the lowest nematode reproduction agrees with observations of many authors who compared reproduction of various nematodes on seedlings or roots in culture, with the same plant or organ on a callus inducing medium (16, 18, 25, 50). Higher P_. penetrans reproduction on host tissues cultured on R medium in comparison to MS medium (Table 1.1) indicated that nematode population increases regardless of increase of tissue fresh weight. This lack of correlation between nematode reproduction and tissue fresh weight agrees with data reported by Krusberg and Blickenstaff (27) , Schroeder and Jenkins (50) and Mitsui (31). Riedel et al. medium (42) is a very simple and practical medium that has been demonstrated to be suitable for maintenance and prolifer­ ation of several Pratylenchus species. Effect of varying concentrations of sugar, 2,4-D and yeast extract on this four component medium on P. penetrans reproduction has been studied (43). However, only the effect of relatively high yeast extract concentrations, varying from 1 to 15 g/1 were studied. As yeast extract is known to have an effect on plant cells similar to plant growth substances, accelerating chromosome 39 mutation, for instance, in a manner comparable to small amounts of kine- tin (53, 54), it was thought to be of interest to study the effect of low rates of this component on P. penetrans reproduction. Indeed, yeast extract even at the low rate of 0.5 g/1 proved to promote nematode pro­ duction comparable to 1 g/1 up to the 80-day extraction for both experi­ ments (Table 1.2 and 1.3). The fact that at 110-days reproduction in medium with 0.5 g/1 yeast extract (Table 1.3) was significantly lower than in 1 g/1 medium indicates that in longer term cultures yeast extract may become a limiting factor. Unfavorable conditions for reproduction on R medium depleted of yeast extract (Table 1.2) did not alter the ratio female/male which was close to 1 (Table 1.4), although host nutrition has been pointed out as the probable factor affecting sex ratio on nematode populations (59, 60). Under conditions of nutrient deficiency male development prevails, resulting partially from sex reversed females (60). However, Maclure and Viglierchio (30) studying the influence of host nutrition on sex ratio of Meloidogyne incognita on excised plants, observed that, even under conditions of extreme crowding, sex ratio of M. incognita was only slightly affected, whereas population development was severely reduced. They concluded that in case of excised plants, which are in contact with a nutrient medium, and therefore not dependent upon translocation of organic compounds from the shoot, nutrient depletion should be less likely to lead to sex imbalance. This could be the reason why sex ratio was not significantly altered throughout this experiment. When large amounts of sterile nematodes are desirable, the cultures are usually incubated for periods ranging from 70 to 140 days before 40 inoculum is used, or before a new subculture is needed in order to increase the amount of inoculum for a future use. Based on results con­ tained in Table 1.7, one would consider the use of French square bottles for £. penetrans culturing which produced cultures with the highest population levels. However, some other points should be taken into con­ sideration for each container. Since French square bottles will hold no more than three alfalfa seedlings each, for instance, they are not too practical for subculture, and they are hard to empty when inoculum is needed. On the other hand, they are the most space efficient container X 7 tested with about 12,800 bottles/m fitting in 1 m of incubator space. Tubes will take 8 to 12 seedlings, they are easy to subculture and to empty, and they have a space efficiency of ca. 6,040 tubes/m incubator. Petri dishes hold more than 12 seedlings, they are easy to subculture and to empty, as well as being readily available. About 6,890 dishes will fit in 1 m of incubator space. They are, however, subject to dehydration, and water condensation on the walls favors contamination. Long term storage is important to the maintenance of cultures in stock. In this case, need for low frequency of subculture is one of the most desirable characteristics, because subcultures are time consuming and always present a certain risk of contamination. If cultures could be maintained for long periods of time without too much labor, this cer­ tainly would encourage nematologists to maintain nematodes in collection or even to establish, for instance, nematode banks, as has been done for the stem nenatode in Sweden (4, 14, 15), or even, to establish nematode collections at national or international levels. 41 Highest populations of P . penetrans were reached in petri dishes compared to the other containers (Table 1.8). This could be influenced by oxygen dependency of nematodes for developmental processes, although we have no data to substantiate it. Van Gundy and Stolzi (63) studying the influence of different oxygen diffusion rates in soil on the survi­ val, movement and reproduction of Hemieye 1 iophora arenaria, observed that largest populations were found in containers exposed to highest oxygen rates and lowest, correspondingly, in containers which had reduced oxygen diffusion rates. Molting and egg deposition appeared to be particularly vulnerable processes to oxygen deficiency for this spe­ cies. In the present study, when nematode populations in different containers (Table 1.8) are calculated in terms of nematode population density, that is, nematode number per area of container (Table 1.9), petri dishes presented the lowest population densities, 37 and 39 nema- 7 todes/cm , for the 5 and 10-month extractions, respectively. Although no measurements of gas exchange have been conducted, this may serve as evidence that petri dishes provide more aeration for nematode and tissue than the other containers. Nematode density in tubes at 10 months was also low, but this was due to a sharp decrease in reproduction (Table 1.8) probably due to other factors. After 5 months of storage, P. penetrans populations were equivalent in French Square bottles and in tubes (Table 1.8). However, at 10 months nematode number in cultures contained in tubes decreased sharply. This may be because the tubes were placed inside plastic bags to facilitate storage. Studies on respiration rates of P. penetrans have shown res­ piration to be higher in air (0.03% C02) than in total absence of (X^ 42

in the gaseous phase (2, 46). As CO 2 concentration increased from 0.03 to 0.11, respiratory rates dropped significantly, increasing again as

CO 2 concentration was increased to 2% (2). Summarizing, P. penetrans

respiration remained at high rates even in presence of higher CO 2 con­ centrations. As tubes were in plastic bags, oxygen concentrations might have been depleted, causing inhibition of nematode development. Long term storage studies should include maintenance of nematode

cultures in absence of or in low levels of CC^, since CO 2 concentration has been shown to increase respiration of JP. penetrans (2, 46). If nema­ todes in such cultures could enter a state close to cryptobiosis, the problem of long term storage would be solved and cultures could be maintained almost indefinitely, once those incubation conditions were compatible also with host tissue maintenance. A number of reports comparing seedlings or excised roots vised as nematode substrates to those same host tissues cultured on callus induc­ ing medium indicate that nematode reproduction is far better on cal- lused tissues (16, 25, 50) . This was observed also in the present study when P. penetrans cultured on potato, carrot, pea and alfalfa tissue on water agar medium presented very poor reproduction compared to the same tissues cultured on R and MS medium (Table 1.1). P_. penetrans is considered essentially a parasite of root cortex (5, 40, 45, 56, 57). In only one instance it was reported damaging vascular tissues of cabbage roots (1). Root endodermis is usually con­ sidered as responsible in preventing nematode invasion of the stele (5, 40, 45, 56, 57). Histological study of alfalfa roots infected under 43 sterile conditions showed that £. penetrans was found only in the cor­ tex (5), usually lying parallel to the stele (38). Our observations are that on alfalfa cultures nematodes occurred most often (about 701 of the cases) on cortical cells around vascular tissues (Plate III) and in masses on parenchymatous-like cells (Plate VI), and sometimes in xylem cells (Plate VII), As nematodes reproduce much better on callused tissue than on seedlings, it would be expected that a higher proportion of them would be found on callus tissue. This observation, added to the fact that an increase in tissue fresh weight does not necessarily mean an increase in nematode reproduction (Table 1.1), makes it difficult to explain which kind of mechanism is trig­ gered to transform seedlings and excised roots in such exceptionally good substrate for nematode reproduction. Mechanical characteristics of callused tissues may explain some of this. In our alfalfa cultures, non-friable callus, i.e., callus that grows as lumps, was formed. This type of callus was reported to have a greater amount of cell wall polysaccharides and a decreased percentage of cellulose compared to pectic substances and hemicelluloses (21) . On the other hand, pectinases have been extracted from P. penetrans (33). If the level of pectic compounds increased and cellulose level decreased in the alfalfa tissues, in a similar manner observed for Vicia faba cul­ tures (21), nematode penetration and locomotion through the callus cells would be much facilitated. It would be interesting to test this hypo­ thesis. Alfalfa seedlings cultured on R medium still seem to be the most practical combination for reproduction and maintenance of P. penetrans. 44 However, it would be better if a host other than alfalfa could be used. Surface sterilization of alfalfa seeds is time consuming due to the small seed size and high internal bacterial contamination. Callus could be induced on whole seedlings or only excised roots of a new selected host, because it seems that P. penetrans does have preference for feeding around vascular tissue. In any plant host study, particu­ lar attention should be given to the cultivars, since they may present different degrees of susceptibility to P. penetrans. The reason for the presence of nematode masses in alfalfa callus tissue, and the physiological stage of these nematodes should be inves­ tigated. Clarifying this point could help to elucidate the nature of the relationship existent between callus cells and nematodes. Tissue age at the time of inoculation is considered important (64) to nematode reproduction. Optimum age on alfalfa was determined to be 2 weeks (27, 41). Recent work in P. penetrans on alfalfa seedlings showed that penetration is significantly greater into young tissue (2 days), followed by medium-age tissue (10 days) and old tissue (20 days) (38). Besides handling, space efficiency and convenience, efficient gas exchange should be another characteristic considered in the choice of containers. Petri dishes seem to be a good container to obtain high nematode populations (Table 1.8), if condensation on the wdlls could be avoided. For long terra storage petri dishes would also provide high populations (Table 1.8), if dehydration could be avoided. If monoxenic cultures of nematodes could be established on cell suspension cultures, for which there are especially designed equipment to promote high rate 45 of gas exchange between liquid and gas phases of the culture, higher nematode populations could probably be developed. Observations on our stock cultures indicate that numerically, more uniform cultures are obtained using sterile nematode suspension ali­ quots for inoculation, instead of infected callus bits from stock cul­ tures which is the method routinely used for maintenance and inoculum production. However, as the use of suspension involves an extra step, it might be better that this inoculation method be reserved for speci­ fic cases where nematode quantification is critical. Routine work of culture maintenance is more easily done using infected callus bits. Based on the data obtained for P. penetrans reproduction (Tables 1.2, 1.3, 2.1 and 2.2) it is difficult to predict what would be the most suitable number of nematodes to be used as initial inoculum to yield high nematode populations. Many factors have to be taken into account. Firstly, nematodes may not survive the trauma caused by the extrac­ tion, and inoculation may provide an overestimation of the initial inoc­ ulum. Townshend (55), for instance, observed that females dissected from alfalfa roots, although without suffering any physical injury, failed to penetrate roots and died 72 hr later, apparently of shock. Thus, potentially inactive nematodes may be considered as initial inoc­ ulum. Secondly, inoculum quality seems to be as important, or perhaps more so than quantity (12, 51, 55). Kable and Mai (23) suggested that only fourth stage and adults of P. penetrans are invasive stages. Sontirat and Chapman (51) partially confirmed those results when 46 studying penetration by females, males and second stage larvae. Pene­ tration of second stage larva was significantly lower. Townshend (55) observed that females penetrated roots earlier and faster than male and third stage. About 80% of the females, 43% of the males and 30% of larvae penetrated alfalfa roots after 6 hr. Olthof (38) found that female and third stage larvae entered roots 122 and 83% more than males, respectively. When only P. penetrans adults were used as inoculum on red clover, 75 to 90% of inoculum penetrated the roots (19). Those results indicated that higher populations may be reached if only adults and fourth stage larvae are used as inoculum. Increasing nematode densities up to 20 nematodes per alfalfa seed­ ling (55) or up to 40 on red clover (19) did not affect penetration. lVhen inoculum level on alfalfa seedling was increased from 25 up to 1,000, nematode penetration increased arithmetically (12). Concluding, a systematic study of several p. penetrans populations should be initiated to verify whether different populations require dif­ ferent culturing conditions. It would not be surprising if this hap­ pens, because monoxenic cultures of P. penetrans originating from a few sources have been used for research work among nematologists. CHAPTER II

Comparative reproduction of four species of Pratylenchus on alfalfa culture

INTRODUCTION AND LITERATURE REVIEW In 1977, Krusberg and Babineau (26) reported that eight species of Pratylenchus had been cultured monoxenically in alfalfa callus. Sub­ sequently, six additional species, P. scribneri (47) , P. pinguicaudatus (6), P. crenatus and £. loosi (32), P. convallariae (31) and P. agilis (L. R. Krusberg, University of Maryland, personal communication) have been successfully cultured in alfalfa callus. Although all 14 species of Pratylenchus have been cultured on alfalfa callus, there is increasing evidence that this might not be the best substrate for all Pratylenchus species. In studying the effects of temperature and pH on propagation of seven Pratylenchus species, Mitsui et al. (32) had to double the period of culture for P. crenatus in order to compare reproductive rates with other species. In a sub­ sequent study Mitsui (31) compared the effects of different media on reproduction of eight Pratylenchus species in alfalfa callus. He obtained considerably lower final-initial population ratios for P. crenatus and P . convallariae in comparison to the other species. Mitsui's data comparing species were offerred without statistical

47 48 treatment, however. Recently, Kimpinski and Willis (24) reported that alfalfa was a poor host for P. crenatus. In our laboratory, observa­ tions on the Pratylenchus species in culture have also indicated dif­ ferential reproduction rates in alfalfa callus. Compared to P_. scrib- neri, £. penetrans and J?. brachyurus, P. crenatus reproduced poorly on alfalfa callus at 20 C and P. agilis barely maintained itself in cul­ ture. Such behavior is not surprising in light of the host range reports for some species. Even though most species of the genus Pratylenchus are highly polyphagous, the literature indicates that some species have a more limited host range. For instance, £. crenatus is reported to damage grasses and cereals, such as oats, barley, rye, and reproduce poorly on potato (20, 29, 39). P. pinguicaudatus is known to attack only cereals (7); P. convallariae has been reported only from rhizomes of Convallaria (7, 29), and P. goodeyi is a specific pathogen of banana (28). Because of reports on restricted host ranges for some Pratylenchus species, the following study was undertaken with two main objectives. The first objective was to quantitatively compare reproductive rates of Pratylenchus penetrans, P. brachyurus, P. scribneri and P_. crenatus cultured on alfalfa callus. This would determine whether further studies are needed to test alternative tissues for improved culture of these species. The second objective was to establish when maximum nematode populations occurred in culture in order to determine optimal time for subculture and/or use of the cultures as inoculum sources. MATERIALS AND METHODS

A. Experimental Plan

The basic replicate in the design was a 30 ml French square bottle filled with IS ml of Riedel et al^. medium (R) (43) (Appendix, Table 3.1) and containing three 'Ranger1 alfalfa seedlings each. Each treat­ ment consisted of 10 replicates. Throughout the experiment a complete­ ly randomized design was maintained. The experiment was done three times.

B . Inoculum Source

The origin of the stock cultures used in this study are as fol­ lows : 1) P. penetrans was obtained from Dr. W. F. Mai, Cornell Univer­ sity, and originated from a single female isolated from apple roots in New York. 2) P. crenatus and P. scribneri were isolated from 'Superior1 potato roots from fields in Columbiana and Seneca County, Ohio, respec­ tively. Cultures of P^. crenatus were started from a single female and cultures of P. scribneri from three to five females. 3) P. brachyurus was obtained from Dr. L. R. Krusberg, University of Maryland. They were extracted from Florida peanut roots.

49 50 All four species were maintained on alfalfa callus at 20 C, using methods of Riedel and Foster (44). They were subcultured every three months.

C. Sterilization of Alfalfa Seeds and Bioassay for Contamination

Alfalfa seeds (Medicago sativa L. 'Ranger') were immersed in con­ centrated sulrfuric acid (36N) for 15 min (36N), rinsed three times in sterile, double distilled water; immersed in 1:1000 mercuric chloride solution in 30% ethanol for 15 min, and rinsed three times in sterile double distilled water. To bioassay for contamination, seeds were placed to germinate on potato dextrose agar for seven days under natu­ ral day-night light conditions.

D. Extraction and Inoculation of Nematodes from the Stock Cultures

To obtain aseptic inoculum, Baermann funnels made from 23 cm dia­ meter pie pans were autoclaved inside paper bags at 1 atm (121 C) for 20 min. Nematodes were extracted aseptically for 48 hr from the stock cultures in a laminar airflow cabinet. The resulting aseptic nematode suspension was dispensed into the French square bottles using a Finn- p pipette (KY Finnpipette, Helsinki). During inoculation the suspen­ sion was continually stirred using a magnetic stirring bar. The volume of the suspension added using the Finnpipette was determined according to the nematode level desired. After inoculating all the French squares of one species, 10 additional aliquots were counted to determine the initial population levels. 51 Inoculation was done seven days after the alfalfa seedlings were incubated on R medium.

E. Incubation Conditions

The French square bottles were incubated at 25+2 C, in the dark.

F. Extraction, Counting and Data Analysis

Nematodes from each container were extracted for 24 hr using modi­ fied Baermann funnels made from 250 ml styrofoam cups and cheese-cloth (Chapter I, Plate I). Tissue and agar medium from each container were extracted sepa­ rately and the suspensions mixed prior to counting. IWo, 5 ml aliquots from the nematode suspension of each species were counted at 40X magni­ fication to determine the total nematode number in each French square bottle. Data obtained were transformed to log x or log x+1, prior to analysis of variance. Treatment mean comparisons were conducted using Duncan's multiple range test. Regression analyses were used to relate nematode reproduction to time. RESULTS

Reproduction of Pratylenchus brachyurus, P. crenatus, £. penetrans and £. scribneri on alfalfa callus at 25 C for 100 and 120-day incuba­ tion periods is shown in Tables 2.1 and 2.2, respectively. P. scrib­ neri showed the highest level of reproduction, with 9868 and 5496 nema­ todes for 100 and 120-day periods, respectively, and P. crenatus the lowest in both periods studied, with 256 and 485 nematodes for 100 and 120-day periods, respectively. P. penetrans and P. brachyurus had intermediate population levels. Reproduction of P. brachyurus across the 100 and 120-day periods was adequately described by the quadratic equations, Y = 2.167 + 0.123X - 0.000744 X2 with r2 = 0.9593 (Figure 2.1-A) and Y = 0.4992 + 0.05404 X - 0.000304 X2, with r2 = 0.9848 (Figure 2.1-B), respectively. Maximum levels of nematodes in culture occurred at 83 and 89 days for the 100 and 120-day periods studied, respectively. Reproduction of P. crenatus across the 100 and 120-day periods was described by the equations Y = 0.9516 + 0.014295 X with r2 = 0.9614 (Figure 2.2-A) and Y = 1.0764 + Q.01248X with r2 = 0.8543 (Figure 2.2-B), respectively. Reproduction of P. penetrans populations in the 100 and 120 days of incubation fit a quadratic model, Y = -0 . 530 + 0.09085X - 0.000584X2, with r2 = 0.9919 (Figure 2.3-A) and Y = 0.82938 + 0.046785X -

52 0.0002348X2, with r2 = 0.9795 (Figure 2.3-B), respectively. Popula­ tions peaked at 78 and 100 days, respectively, for■the cultures studied for 100 and 120 days. Production of P. scribneri against time for the 100 and 120-day periods was well described by the quadratic equations Y = -0.611 + 0.0956X - 0.0005075X2, with r2 = 0.9960 (Figure 2.4-A) and Y = -0.8648 + 0.099315X - 0.0005395X2, with r2 = 0.9935 (Figure 2.4-B), respective­ ly. Maximum populations occurred at 94 and 92 days, respectively, for the periods of 100 and 120-day incubation. 54

Table 2.1. Reproduction of four Pratylenchus species on alfalfa callus cultured on Riedel's et al. medium at 25C for 100 days.

Nematodes/10 cultures* Initial Days after inoculation Inoculum 4U 60 8ff roo

P. brachyurus 3 9 V 56Zc 670 b 784 b 748 b p. crenatus 52 b 37 c 92 c 156 c 256 c p. penetrans 39 c 186 b 716 b 1484 b 576 b p. scribneri 59 a 331 a 4828 a 7236 a 9868 a x Each culture consisted of callus from three alfalfa seedlings, y Values are the mean number of nematodes in ten inoculation aliquots. 2 Values in a column followed by the same letter are not significantly different according to Duncan’s multiple range test at P = 0.01. Data were transformed to log x prior to analysis. 55

Table 2.2. Reproduction of four Pratylenchus species on alfalfa callus cultured on Riedel's et al. medium at 25C for 120 days.

Nematodes/10 culturesx Initial Days after inoculation Inoculum 40 60 80 100 120

P. brachyurus 102yaz 219 a 579 b 838 c 794 c 523 c p. crenatus 81 b 45 b 83 c 230 d 229 d 485 c p. penetrans 84 b 241 a 904 b 1614 b 1390 b 1338 b p. scribneri 53 c 247 a 2308 a 5496 a 5493 a 2982 a x Each culture consisted of callus from three alfalfa seedlings, y Values are the mean number of nematodes in ten inoculation aliquots, z Values in a column followed by the same letter are not significantly different according to Duncan1 s multiple range test at P = 0.01. Data were transformed to log x prior to analysis. 120 56 100 80 60 days of incubation UO r2 = 0.9593 = r2 rZ = 0.9848 = rZ Y = -2.167 + 0.123X - 0.000744X 0.000744X - 0.123X + -2.167 = Y Y = 0.4992 + 0.05404X - 0.000304X 0.000304X - 0.05404X + 0.4992 = Y 20 * Each point is the mean of 10 replications. 10 of mean the is point Each * 120-day period. 120-day medium at 25 C. C. 25 at medium A) period; 100-day B) a for a for 0 10 10 100 100 1,000 l.OOC 10,000 10,000 Figure 2.1. Reproduction of Pratylenchus brachyurus on Riedel's et al. et Riedel's on brachyurus Pratylenchus of 2.1. Figure Reproduction

nematodes/container nematodes/container 120 57 100 8o 60 days of incubation r2 = 0.8543 = r2 Y = 1.0764 + 0.01248X 0.01248X + 1.0764 = Y r2 = 0.9604 = r2 Y = 0.9516 + 0.014295X 0.014295X + 0.9516 = Y 20 120:-day period. 120:-day * Each point is the mean of 10 replications. 10 of mean the is point Each * medium at 25 C. C. 25 at medium A) period; 100-day a B) for a for 0 10 10 100 100 1,000 1,000 10,000 10,000 Figure 2.2. Reproduction of Pratylenchus crenatus on Riedel's et al. et Riedel's on crenatus Pratylenchus of 2.2. Figure Reproduction nematodes/container nematodes/container 120 58 M 100 8o 60 days of incubation Y = 0.82938 + 0.046785X - 0.0002348X 0.0002348X - 0.046785X + 0.82938 = Y r2 = 0.9795 = r2 r2 = 0.9919 = r2 Y = -0.530 + 0.09085X - 0.000584X 0.000584X - 0.09085X + -0.530 = Y 20 * Each point is the mean of 10 replications. 10 of mean the is point Each * period. medium at 25 C. A) for a 100-aay period; B) for a TZO^Say TZO^Say a for B) period; C. 25 at 100-aay a A) medium for 0 10 10 100 100 1,000 1,000 10,000 10,000 Figure 2.3. Reproduction of Pratylenchus penetrans on Riedel's et al. et Riedel's on penetrans Pratylenchus of 2.3. Figure Reproduction nematodes/container nematodes/container 120 59 100 80 60 days of incubation r2 = 0.9935 = r2 Y = -0.8648 + 0.099315X - 0.0005395X 0.0005395X - 0.099315X + -0.8648 = Y r2 = 0.9960 = r2 Y = -0.611 + 0.0956X - 0.0005075X 0.0005075X - 0.0956X + -0.611 Y = 20 * Each point is the mean of 10 replications. 10 of mean the is point Each * day period. day medium at 25 C. C. 25 at medium A) period; a 100-day B) for T2*0- a for 0 10 10 100 10G 1,000 1,000 . 10,000 10,000

nematodes/container nematodes/container al. et Riedel's on scribneri Pratylenchus of 2.4. Figure Reproduction DISCUSSION

Results of this study indicate that Pratylenchus brachyurus, P. crenatus, P_. penetrans and P. scribneri reproduced differently on alfalfa callus when maintained under similar conditions. Initial ino­ culum levels did not seem to significantly affect reproduction levels for the species during both periods studied (Tables 2.1 and 2.2). For example, P. scribneri started the 100-day period with the highest initial inoculum levels of the four species (Table 2.1), and the 120- day period with the lowest initial inoculum (Table 2.2). However, independent of inoculum levels, P. scribneri developed the highest populations in both experiments. In the case of P. brachyurus, although the initial inoculum was high for the 120-day period (Table 2.2), population levels did not indicate any advantage for that species as compared to the other species. Olowe and Corbett (37), adding from 0 to 1,000 P. brachyurus to excised maize roots, obtained maximum pene­ tration at ca. 250 nematodes. Above this level penetration decreased. The authors did not describe the method of inoculation used. Although no indication was found in the present study that the initial inoculum played an important role within the range of inoculum values used, data relative to influence of initial inoculum on nematode population should be looked at with some reservation, because there is always the possibility of nematode death as a consequence of extraction and

60 61 inoculation procedures. Townshend (55), for instance, inoculating alfalfa seedlings with single females dissected from infected seed- lings, observed failure in penetration and death of 80% of these females 72 hr after inoculation. In the case of P. brachyurus and P. scribneri, estimated values for maximum population development were very close, for both periods studied. P. brachyurus reached maximum numbers at 83 and 89 days, and P. scribneri at 94 and 92 days, for test periods of 100 and 120 days, respectively. However, in the case of P. penetrans maximum population developed in 78 and 100 days during culture periods of 100 and 120 days, respectively. Results of two other experiments conducted with P. -penetrans under the same conditions (Chapter I, Figures 1.1-A and 1.2-B) showed 80 and 86 days as the time for development of maximum populations, with both populations derived from the same initial ino­ culum, estimated at 67 nematodes per bottle (Chapter I, Table 1.3). This variability found in the present study may have resulted from the bisexuality of £. penetrans. The quality of the initial inoculum (12, 51, 55) or proportion of larvae, males and females, may have influenced the time when maximum populations developed. The other three species, being parthenogenic, may have been unaffected by this. Reproduction of P_. crenatus was significantly lower and slower compared to the other species (Table 2.1 and 2.2). At 120 days the populations were still increasing (Table 2.2, Figure 2.1). Even so, the maximum population reached in this period, 485 nematodes (Table 2.2), was significantly lower when compared to maximum populations of the other species. This slower development agrees with observations 62 of other authors (32), who had to extend the time of P. crenatus in culture in order to compare this species with others in a temperature study. Temperature is known to be an important factor affecting nematode movement and rate of growth and reproduction, and it is considered par­ ticularly important for Pratylenchus (29). Even geographic distribu­ tion of species pertaining to this genus seems to be influenced by tem­ perature (58). The optimal temperature for reproduction of P. crena­ tus is 10 to 15 C (8). Kimpinski and Willis (24) working with alfalfa and timothy in incubators found that P_. crenatus on alfalfa decreased in number as temperature increased from 10 to 27 C, and increased on timothy as temperature rose from 10 to 27 C. These results do not agree with Mitsui's at al. (32), who reported highest reproduction of P. crenatus on alfalfa callus at 25 C, in a temperature study ranging from 15 to 30 C. As alfalfa seedlings cultured on callus inducing medium are quite different from the original seedlings, it may be that optimal temperature for reproduction depends more on nematode-host interaction than on the nematode itself, as pointed out by Dickerson (11). However, we have been observing in our cultures that P. crenatus does seem to do better at 20 C than at 25 C. It would be interesting to compare temperature requirements and also, to look for morphological differences between our P_. crenatus population isolated from ’Superior1 potato roots in Columbiana County, Ohio, and the one studied by Mitsui et al. (32) isolated from pasture in Hokkaido, Japan. Different geo­ graphical races could have different temperature requirements. 63 In conclusion, more studies are needed to optimize culture tech­ niques for P. crenatus and P. agilis. Further studies should focus on alternative tissue/temperature combinations. P. crenatus is known to be a cereal pathogen (8), and high populations have been reported to develop on oat, rye and barley roots (10). However, our experience is that the use of small grains for the systematic establishment of mono- xenic cultures is quite impractical. Because of seed characteristics of small grains, husks and highly hydrophobic seed coat, efficiency of surface sterilization treatments is very low and, consequently, con­ tamination levels may reach as much as 90%. A possibility for the use of those grains as nematode substrate would be to maintain stock cul­ tures of excised roots on medium without hormone and, as roots grow, to periodically transfer root explants to callus inducing medium. Initial inoculum values in the range studied did not seem to influence final population. However, there is in the literature some indication that Pratylenchus penetration is affected by inoculum level. For example, populations of P. zeae or P. brachyurus greater than ca. 250 nematodes inhibited penetration of maize roots by these species (39). For P. penetrans penetration increased with increased popula­ tions from 25 to 1,000 nematodes per alfalfa seedlings (12). However, up to 200 nematodes, percentage concentration was very low (12). It is possible that each species has a different critical number of maximum inoculum level, which will also depend on plant host used as substrate. With exception of P. crenatus, other Pratylenchus species studied developed reasonable populations on alfalfa seedings cultured on R medium. 64 According to the estimated times for maximum populations for each species, subculture of stock cultures should be done each three months for brachyurus and P. scribneri, between each three to four months for P. penetrans, and each four months for _P. crenatus. Since nematode population development differed for the four species studied, to obtain equivalent final populations for mass pro­ duction of inoculum cultures should start with different amounts of stock cultures. P. scribneri, for example could start with about half of the initial amount of stock cultures used to start cultures of P. brachyurus and P. penetrans. P_. crenatus will require at least three times more. No doubt as further Pratylenchus species are brought into culture, optimal tissue/temperature combinations must be developed. BIBLIOGRAPHY

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Composition of Riedel1s et al. and Murashige and Skoog media

71 72 « Table 3,1. Composition of Riedel’s et al. medium (1973).

Components g/1 1^0 yeast extract 1. sucrose 20. agar 10. 2,4-D 0.002 73 Table 3.2. Composition of the Murashige and Skoog's medium (1962).

Components mg/1 H20

nh4no 3 1650

kno3 1900 CaCl2.2H20 440 MgS04.7H20 370

k h 2po4 170 Na2EDTA. 37.3 FeS04.7H20 27.8 H3B°4 6.2 MnS04.4H20 22.3 ZnS04 .4H20 8.6 KI 0.83 Na^4o04. 2H20 0.25

Cu S04.5H20 0.025 CoCl2.6H20 0.025

myo-inosotol 100 nicotinic acid 0.5 pydrioxin-HCl 0.5 thiamin-HCl 0.4

sucrose 20. agar 10.