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Xerox University Microfilms 300 North Zaab Road Ann Arbor, Michigan 4S106 76-24,671 RAO, Balakrishna, 1944- PYTHIUM ROOT ROT OF CORN: PYTHIUM GRAMINICOLA AND OTHER CAUSAL AGENTS INVOLVED; DETECTION OF P. GRAMINICOLA IN SOIL; AND EFFECTS OF TILLSGE^ ROTATION. FUNGICIDES, MOISTURE, AND TEMPERATURE. The Ohio State University, Ph.D., 1976 Agriculture, plant pathology
Xerox University Microfilms,AnnArt»r,Michigan4etoe PYTHIUM ROOT ROT OF CORN: PYTHIUM GRAMINICOLA AND OTHER CAUSAL AGENTS
INVOLVED; DETECTION OF P. GRAMINICOLA IN SOIL; AND EFFECTS OF TILLAGE*
ROTATION, FUNGICIDES, MOISTURE, AND TEMPERATURE.
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By Balakrishna Rao, B.Sc., M.Sc., M.S.
* * * * *
The Ohio State University *
1976
Reading Committee: Approved By
Dr. A. F. Schraitthennor
Dr, C. W. Eliott Co-Adviser, Department of Plant Pathology
Dr. L. E, Williams 0 ^
Co -Advi ser, Dcparfyent of Plant Pathology ACKNOWLEDGEMENTS
Tho author thanks his advisors Dr. A. P. Schmitthenner and
Dr. C. W. Ellett for their guidance, encouragement, and constructive criticism during the course of this research and throught his graduate caroer. He wishes to express his appreciation to Dr. R. W. Caldwell for his suggestions, assistance and constructive criticism, and to
Dr. L. E. Williams for reviewing the disscrtaion and offering helpful criticism. He is indebted to Dr. I. W. Deep and Dr. L. E. Williams for providing financial assistance and facilities. Thanks aro also extended to Dr. D. M. Van Doren for providing field plot facilities, to Dr. C. R.
Weaver for statistical assistance, to Dr. G. Louie for providing seeds, and to Mr. G. Borkey for assistance with photography. Appreciation Is extended to staff members, graduate students, and technicians of the
Department of Plant Pathology at The Ohio State University and The Ohio
Agricultural Research, and Development Center. The author would like to thank one and all tho members of his and Borchers family for their encouragement and assistance throughout this investigation.
ii VITA
July 23, 1944 ... Born - Mysore, India.
1964 ...... B.Sc., University of Mysoro,'India.
1964-1966 ...... M.Sc., University of Bombay, India. Teaching. Merit scholarship recipient.
1966-1967 ...... Teaching, University of Bombay, India.
1967-1968 ...... Post M.Sc Diploma, Univorsity of Madras, India. UGC (University Grant Commission) scholarship recipient.
1969-1971 ... M.S., (Mycology), Univorsity of North Carolina. Teaching and Research Assistant.
1971-1973 ...... Worked on Biological control of Mosquitoes of medical importance with Dr. J. N. Couch, Univ orsity of North Carolina (NIH funded proj ect).
1973-1976 ...... Graduate Research Associate, Department of Plant Pathology, The Ohio State University and The Ohio Agricultural Research and Development Center, Wooster, Ohio.
PUBLICATIONS
1. Rao,B., and W. J. Koch. 1970.' Fungal survey in waste ponds and c .• creek, Jta II. T. Odum and A. F. Chestnut cd. Studies of marine estuarino ecosystems developing with treated sewage wastos. Annual Report. 1969-1970. Univ. of North Carolina, Chapol Hill, '311-317 p.
2. Couch, J. N., S. V. Romney, and B. Rao. 1974. A new fungus which attacks mosquitoes and related Diptera. Mycologia 6 6 :374-3 1 7 .
FIELD OF STUDY
Major field: Plant Pathology.
iii / TABLE OF CONTENTS
Page ACKNOWLEDGEMENTS...... il vita ...... m .
LIST OF TABLES vi l i s t o f f i g u r e s ...... viii
INTRODUCTION ...... 1
PART I: PYTHIUM ROOT ROT OF CORN: PYTHIUM GRAMINICOLA AND OTHER CAUSAL AGENTS INVfjLVtUTAND THEIR VIRULENCE
Introduction 5
Materials and Methods...... 14
Results...... 19
Discussion ...... 32
PART II: PYTHIUM ROOT ROT OF CORN: METHODS FOR DETECTION OP PYTHIUM GRAMINICOLA IN SOIL
Introduction '...... 37
Materials end Mothods...... 40
Results ...... 43
Discussion . . . ; ...... ; ; 1 ; . . . . 52
*
iv Page PART III: PYTHIUM ROOT ROT OP CORN: EFFECTS OF TILLAGE, ROTATION, FUNGICIDES, MOISTURE AND TEMPERATURE
Introduction ...... 56
Materials and Methods ...... 60
Results......
Discussion ...... 9*
SUMMARY...... 98
LITERATURE CITED...... 103
V LIST OF TABLES
Tabic Number Page
1. Percentage of corn root lesions from which fungi wore isolated from field plots at bi-weekly intervals during 1974 and 1 9 7 S ...... 23
2. Bi-weekly summer precipitation at tho North Central Branch, 1974, and the North Westorn Branch, 1975 OARDC. . 25
3. Fungi recovered on SAPBNC media at monthly intervals from roots of com. growing in tho greenhouse in field infested s o i l ...... 27
4. Relative virulence of Pythium spp. on corn seedlings using the petri dish water culture seedling test. .... 29
5. Number of surviving seedlings of two varieties of crested wheatgrass two weeks after planting in soil from tillage plots ...... 45
6. Mean number of total lesions and small lesions on corn seedlings roots suspended in water ovor throe different volumes of soil (CRT bioassay) collected at five sampling d a t e s ...... 48
7. Mean lesion score and number of large lesions on corn seedling roots suspended in water over three different volumes of soil (CRT bioassay) collected at fivo sampling d a t e s ...... 49
8. Mean lesion number, lesion score, small lesions and large lesions on corn seedling roots suspended in water over three different volumes of soil (CRT bioassay) collected from two tillage systems on Toledo silty dlay s o i l ...... 51
9. Interaction of time with tillage and time with rotation for porcent root rot damage in1975 in Hoytville soil . . 72
10. Percentage root damage in the best and worst tillage- rotation, fungicide treatments (TRF) in field plots in corn at different sampling tim e s ...... 74
vi Tabic Number Page
11. Numbers of small (type 1 and 2) lesions, large (typo 3, 4 and 5) lesions and percentage damage of corn seedling roots used for assaying Pythium levels in field soil at seven times during the growing season . . . 77
12. The effect of crop rotation on percent Toot damage in c o m havestcd at three different times in a greenhouse t e s t ...... gj
13. The effect of fungicides on percent root damage in c o m harvested at three different times in a greenhouse test . 82
14. The effect of moisture on percent root damage in c o m harvested at three different times in a greenhouse test . 83
15. Effect of moisture on number of small and largelesions on corn roots at three sampling times ...... 85
16. Interaction of moisture, fungicides and time of isolation on recovery of Pythium gramirticOla and Fusarium spp. from disoased roots in the greenhouse . . . 87
17. Interaction of temperature and moisture on number of infected corn roots in growth chamber t e s t s...... 90
vii LIST OF FIGURES
Figure Number Page
1. Corn root system with root rot symptoms ...... 20
2. Root rot rating* lesion types (small* large* streaked* girdled and necrotic) ...... 21
3. Percentage of corn root lesions from which fungi were recovered in 1975 ...... 26
4. Crested whcatgrass seedling bioassay (CWG) method to detect levels of Pythium spp. in infested soil (tilled and nontillcd soil) ...... 44
5. Corn seedling root trap bioassay (CRT) method to detect levels of Pythium spp. in infested soil .... 47
6. Mean percentage root rot damage of corn in field plots over all tillage, rotation* and fungicide treatments during seven sampling periods in 1975 ...... 69
7. Mean root weight of corn from field plots over all tillago* rotation and fungicide treatments during seven sampling periods in 1975 70
8. Field infected root systems with differences in root mass from tillage, rotation and Pyroxychlor treated p l o t s ...... 75
9. Root samples obtained from greenhouse test showing differences in root mass and discoloration ...... 80
10. Root samples from growth chamber experiment showing differences in root mass and discoloration ...... 89
viii INTRODUCTION
In Ohio the 1975 c o m crop was valued at 787 million dollars.
Disease losses are estimated at 12-15%. Stalk, ear and root rots probably account for the greater amount of loss ( C. W. Eliott, personal communication ). Root rot of c o m has not been given proper attention in recent years. Dickson (14) discusses corn root rot caused by Pythium graminicola Subr. and £. arrhenomanes Drochsler. Ho suggosts root rot may be implicated in tho poor yiolds of corn in certain crop sequence and tillago systems. However, the rccontly publishod'tompendium of Corn
Disoasc^'(77) did not discuss oithor c o m root rot or mention £. gramini cola although corn seedling blights caused by other Pythium spp. are discussed. On a poorly drained soil in Ohio c o m yields were reduced 13% resulting from continuous cropping to corn without tillage (99). Similar yield depressions in continuous no-till corn in poorly drained soils has been reported elsewhere ( 22, 23 ). It has been established that corn yiolds were negatively correlated with corn root rot (25). Best yields and least root rot were obtained in corn following soybeans and lowest yields and most root rot were present, in continuous corn (109). However, it was not established if the rotation effect was the result of Pythium root rot or if there was any effect of rotation m levels of IP. graminicola
Johann et al. (41) reported that in Illinois, Pythium caused a 31% reduction in average seedling stand and a 65% reduction in yield.
1 Root rot of corn may bo particularly important because of the rela
tionship of root damage and stalk rot (109). Whitney and Mortimore (108) reported that stalk rot of corn in Southwestern Ontario began .as a root rot that progressed into the stalk. Tho causal agent, however, was not identified. Other workers (36 , 50, 53 , 73) have noted an association between root necrosis and stalk rot in corn. McKeen (50) has described a stalk rot of corn in which tho disease symptoms first appeared as rot of the root system that extended into the stalk. A bacterium was isola ted from the upper edge of the rotted area pf the' diseased ' stalks; ‘
.P. arrhenomanes was located in a brownish area below this and finally a
Fusarium sp. was located in a reddish zone in the lowest part of the rotted area. Wall and Mortimore (102) reported that root rot preceded stalk rot by 3-5 days. Studies of root rot of c o m (108) indicated that it was a rapid breakdown of senescent parenchymatous tissue in roots and lowor stalks. Craig and Hooker (13) reported that Diplodia zeae (Schw.)
Lew., which causes stalk rot, progressed into the stalk by way of infected roots.
Mwanza and Williams (57) suggested that maize dwarf mosaic virus
(MDMV) increased the susceptibility of corn to several pathogens. They also found that virus-infected, greenhouse grown c o m and wheat were more susceptible to root rot, stalk rot and seedling blight due to
P. graminicola, Diplodia zeae, Fusarium moniliforme (Sheld.) Snyd. and
Hans., F. oxysporum (Schlecht.) Snyd. 8 Hans., F_. roseum and Helmintho- sporium pedicillatum Henry than virus free corn. Tu and Ford (96) demonstrated that corn seedlings infected with MDMV were more susceptible to root rot diseases incited by Gibberella zeae and H» pedicillatum than than were virus-free ones. 3
Root rot with browning and root tip necrosis is a widespread dis
ease of corn. Many investigators have reported the occurrence and
association of different organisms with root rot. However, the earlier
literature concerning the origin and dvelopment of corn discasos did
not always distinguish bcween root rot, stalk rot and oar rot. Root
rot is dependent upon several factors and agents atid is a distinct
disease phase from c o m stalk and ear rot. Root rot diseases cause
major losses in corn production (5).
Because of the importance of corn root rot and the possible rela
tionship of root and stalk rot resulting in great potential yield loss,
and because the cause and etiology of corn root rot is not clearly understood, this investigation was undertaken. Major emphasis was placed bn determining the primary causal agents, developing methods for detect ing lovels of tho primary causal agents in soil, investigating the effects of cultural and environmental factors which might influence tho severity of corn root damage and applying systemic fungicides for control of root rot. paiit i
PYTHIUM ROOT ROT OF CORN: PYTHIUM GRAMINICOLA AND OTHER
CAUSAL AGENTS INVOLVED AND THEIR VIRULENCE.
4 INTRODUCTION
Corn root rot has como to be known over the corn belt as a serious
diseaso of corn in tho midwestem United States. Historically, the
causes of root rot have been diagnosed differently by different people.
Branstetter (5) and Manns (47) have summarized the early literature on corn root rot. Selby (72) reported that root rot of corn was wide spread throughout Ohio in 1918, Hoffer et al. (33), Branstetter (6),
Trost (92), Holbort ot al. (35) and Koehler et al. (43) believed that corn root rot was caused by Gibberella saubinetii (Mont.) Sacc.,
Diplodia zeae (Schw.) Lew., Fusarium moniliforme (Sheld.) Snyd. 8 Hans., and Cophalosporlum acremonium Butler. Clinton (11) reported that the, occurrence of an oospore producing fungus in tho crown region of corn affected with root rot, and he believed it to be Phytophthora cactorum
(Lebert 8 Cohn) Schr. Later an oospore-producing fungus in corn was identified as Pythium ( 5, 41). Hoffer and Carr (32) concluded that corn root rot was duo to mineral difficiency. According to,Hoffer (31) iron compounds accumulate in nodal tissues of corn plants growing in soils deficient in available potassium. The accumulation of iron results in a broakdown • of certain cells in the node which prevents the movement of food from the leaves to the roots causing the roots to become more susceptible to root rotting fungi. To datct,species of Pythium, Pyreno- chaeta, Rhizoctonia, Phialophora, Helminthosporium and Fusarium have been isolated from c o m roots. Each of these will be reviewed separately.
5 Pythium: Pythium spp. wore first isolated from corn roots by Johann
et al. (41), Valleau et al. (97) and Branstetter (5). Drechsler (16)
designated the Pythium of Johann et al» (41) as P. arrhenomanes Drech.
The isolates of Valleau et al. (97) and Branstetter (16) wore similar.
In 1928, Subramanian reported that an isolate of P. graminicola Subr.,
pathogenic on seedling surgarcane in India, was also capable of attacking
barley, maize and sorghum (87). Roldcn (67) identified and reported
JP. arrhonomanos var. phillipinensis as a cause of root rot of corn in
the Phillipincs. Drechsler (IS) concluded that P. graminicola and
P,. arrhenomanes were separate species, but Sprague (82) did not concur.
Melhus et al. (54) in Iowa reported that P. graminicola caused
abundant brown lesions on roots, whoreas P. dobaryanum caused little if
any infection of tho root system. Ho (28) reported both £. graminicola
and dobaryanum pathogenic to corn roots. Most Pythium workers did
not identify the specios with which they wero concerned. Stutzman (86)
suggosted that P. graminicola, presumably,consisted of sovoral patho
genic races that may vary considerably in host range and virulence under different environmental conditions. He also stated that it is not too difficult to conclude that P. arrhenomanes is, in roality, a
highly virulent pathogenic strain of £, graminicola. Rand * and Dopp
(65) found strains of P. arrhenomanes that differed in virulence on
corn and sugarcane. Ho (28), in his isolation of £. graminicola from
corn roots, found that several isolates differed in pathogenicity.
Vanterpool (100) in Canada reported Pythium isolates from wheat which wero similar to P. arrhenomanes. Buchholtz (7) found that P. gramini cola prevented germination, pruned the roots and induced stunting and death of com. Ho and Koepper (29) reported considerable differences 7
in tho response of hybrid seedling c o m to £. graminicola, but found
inconsistency between replicates. Rands and Dopp (65) established that
environmental conditions altered the pathogenicity of a particular
isolate.
More recently Someniuk (73) and Staffoldt (83) also reported Pythium ,
as corn root pathogen. However, emphasis was placed on the role of
Pythium in pre-emcrgonce damping off of com. Hoppo and Middleton (38)
isolated £. voxans do Bary, £. paroecandrum Drech., £. rostratum Butl., ■** ■ £_. ultimum Trow.. £. dobaryanum and.P. irregularo Buis, from decayed
kernels of corn in a numbor of different soils. Hooker (37) reported
that £. dobaryanum and £. splondons Braun wero highly virulent seed
rot pathogens, whereasj £. aphanidermatum (Eds.) Fitzp. and JP. irregu-
larc wore loss virulent. On tho other hand JP. graminicola, JP. paroocan-
drum and P. arrhonomanos causod seedling root rot. £. rostratum and
P. acanthicum Drech. wero avirulent. Erwin and Cameron (19) reported
that £. graminicola caused root rot on five varieties of sweet corn in;a
greenhouse experiment.
Pyrenochaeta; Phoma terrestrls Hanson has been isolated from pink
root rot of corn (45, 39), Johann (39) found it both alone and in combi
nation with Fusarium moniliforme and Pythium. She notod some differences
in susceptibili.ty in root damage dmong single cross hybrids. Kreutzer
(45) reportod £. torrestris was only slightly virulent on corn seedlings.
Carvajal (8) isolated P. terrestris from sugarcane roots in Louisiana, which was pathogenic to corn seedlings in the laboratory. Gorenz et al.
(21) transferred P. terrestris to the genus Pyrenochaeta due to the setose character of tho pycnidium. Craig and Koehler (13) reported that
Pyrenochaeta terrestris (Hansen) Gorenz, Walker and Larson was the only 8
fungus isolated from corn seedling roots that could induce red root
systems. Other fungi isolated wore species of Fusarium, Pcnicilliutn.
Periconia, Phaeocytosporella zeae Stout and Trichoderma.
Rhizoctonia: Sprague reported R. solani Kuhn occurred on c o m in
13 states but gave no details (81). Oswald (S9) found this fungus predominantly in some fields in California and reported that it could cause root rot of barley. Shultz and Bateman (76) found that the emer gence of corn at S C was 76% and 91%, respectively, witli and without
R. solani added to non-sterile soil. Emergence at 25 C was decreased by 51% in tho presenco of R. solani.Ho (28) and Staffeldt (83) consi dered II. solani as a moderately destructive pathogen of corn roots.
Wood (112) isolated R. solani from corn roots during midseason.
Phialophora: Mckeon (51) reported tho occurrence of Phialophora radicicola Cain in rotted corn r6ats .in Canada. " He'suggested that this might havo been mistaken for R. solani. To isolate this fungus, appnrcntly-healthy tissue along with the mycolium adhering to the infected root surface should be used' for plating. The fungus can attack corn roots at any time throughout the season and its brown runner hyphae grow parallel to the roots. The fungus is not very aggressive and apparently is followed by numerous secondary organisms.
Helminthosporium: Shepherd (75) and Shepherd et al. (74) wero the first to report Helminthosporium pedicillatum Henry on the roots of corn.
Henry (27) previously had described this species from root rot of wheat in Minnesota. Tveit (95)’ found H. pedicillatum only slightly pathogenic to four oat varieties tested. Nelson (58) described the perfect stage of this organism as Trlchometasphaeria pedicillatum Nelson. This fungus appears to be rare and perhaps associated with minor root disoases 9
of tho Gramineac. The occurrence and role of H. pedicillatum as a root
rottor is little understood. An unidentified species of Helminthospo
rium caused seedling blight in Wisconsin (85). TU and Ford (96) found
that corn seedlings infected with maize dwarf irtosaic virus (MDMV) were
susceptible to root rot diseases incited by Gibberella zeae and H. podi-
cillatum. Root rot was most severe in MDMV-diseased corn seedlings at
high amounts of fungus inoculum. Mwanza and Williams (57) found that
MDMV- infected corn seedlings wore susceptible to H. pedicillatum. Ho
(28) found H. sativum P., K § B. moderately destructive to corn roots.
Fusarium: Fusarium rosoum f. sp. cerealis (LK.) Snyd. $ Hans,
is a well established pathogen of c o m roots and stalks ( 33,. 103, 104).
A number of workers have contributed to tho understanding of the occurr
ence and biology of this pathogen (46, 103, 104). Fusarium roseum as
it is now constituted, or its form V. roseum f. sp. cerealis, includes
many of tho fusaria commonly associated with root rots and designated
previously under such species names as £. graminearum Schw, £. culmorum
(Smith) Sacc., £. avenaceum (Fr.) Sacc., scirpi Lamb et Fautr,,
£. oquiseti (Cda.) Sacc., and £. sambucinum Fkl. The taxonomic treat ment in tho prosont study is based on Snyder and Hansen (79, 80).
Early workors, Selby (72), Hoffer et al. (33), Branstotter (6),
Trost (92), Holbert et al. (35), Koehler et al. (43), considered that
corn root was caused by Gibberella saubinetii (Mont.) Sacc., Diplodia
zeae (Schw.) Lew and moniliforme (Sheld.) Snyd. 6 Hans. Hoffer et al.
(33) proved that corn root rot and wheat scab were caused by the same
Fusarium sp. and related these with tho perfect stage belonging to the genus Gibberella. Ho (28), concluded that G. saubinetii was a highly
destructive pathogen in Iowa. In the northern United States Gibberella spp. have been mostly associated with stalk and root rot (3), whereas Pythium
and Gibberella have been reported as chief incitants of root rot in
Canada (108). Tho consideration of F. moniliformo as one of the major
causes of stalk rot and seedling blight began with Valleau*s (98) work.
Since that time some workers have advanced evidence supporting Valleau's
findings. There are many reports of F. moniliforme associated with corn
root and stalk rot (72, 98, 97, 47, 66, 46, 59, 6, 33, 35, 39, 43, 61,
60, 92, 64). Ho (28) and Valleau (97) classified £. moniliforme as
slightly destructive to corn roots.
Palmer and Kommedahl (61) isolated Fusarium oxysporum (Schlocht.) '
Snyd. 5 Hans., £. moniliforme and £. roseum from rootworm infested
roots of corn. F. oxysporum was most abundant but grew only on tissues
damaged by tho rootworm. F_. roseum and F. moniliforme were pathogenic
but not isolated frequently. Incidence of roots infected by Fusarium
species incroased as the incidence of root worm infestation increased
in the field.
. Relative virulence of corn root rot pathogens: Richardson (66) reported that Pythium was most virulent, Helminthosporium less virulent and Fusarium least virulent on corn roots. Ho (28) studied the patho genicity of nine fungus species and classified them into three groups;
Those species of fungi capable of being very destructive included
P. debaryanum, P. graminicola and Gibberella saubinetii; those modera tely destructive were Rhizoctonia solani, Holminthosporium sativum,
Diplodia zeae and Penicillium oxalicum; and those slightly destructive wore Aspergillus niger, Fusarium moniliformo, Trichoderma lignorum and
Rhizopus sp, Melhus et al, (54) reported P. graminicola •was more viru lent than P. debaryanum on corn roots. Oswald (59, 60) considered 11
£.• roseum a more important root rot pathogen of corn than JP. oxysporum and JP. moniliformo in California, Warren and Kommedahl (103, 104) isolated P. oxysporum more commonly from corn roots ‘than J?. solani or
£. roseum. Fusarium oxysporum caused some damage on wounded roots,
F^. solani was not pathogenic, and £. roseum was most pathogenic.
From this survey of c o m root pathogens it is concluded that « 9 P_. graminicola (P. arrhenomanes) is tho most virulent followed by
£. roseum, H. pedicillatum, Pyrenochaeta terrestris and other Fusarium spp. There is not enough information on Rhizoctonia solani or
Phialophora radicicola to assess their role. Other Pythium spp. appear to be involved in pre-emergence damping off only.
Seasonal variation in corn root rot; Even though root rot of c o m is a gradual and continuous disease development phenomena, in recent years there has been some indication that it is serious during early
June and late September (25). Ho (28) reported that Pythium dobaryanum attacked vory early but that greator root rot by £. graminicola occurred lator in the season. He found that Pythium damage to barley and corn was much less severe during the summer than in tho spring. Sprague (82) found £. debaryanum. II. sativum and Fusarium spp most commonly on seed lings of cereals and grasses in the early spring. Pythium arrhenomanes caused seed rotting in May, Juno and early fall seedlings. Limber (46) found £. moniliforme abundant during May and early June. Wood (112) reported that P, debryanum was greater during early season and
£.* gra^inicola, Gibberella roseum and R. solani were greater during mid season. Following this succession, several Fusarium spp. and certain other soil-inhabiting fungi, such as H. sativum, Ponicillium oxalicum and Trichoderma spp. were isolated in Ohio. Elliott (18) also studied the fungi associated with corn root rot disease at several stages of root development and found a succession of organisms during the growing season.
Ho, Meredith and Melhus (30) recovered P. graminicola frequently from roots of barley plants in May and Juno, but not in July. Summers and Buchholtz (88) reported considerable variation in occurrence of
P. graminicola on roots of field-grown corn. They found a high freq uency of occurrence in early June (37.5%) and early September (15.8%).
Its absence during late July and early August was associated with low precipitation and high daily soil temperature maxima from Juno 10 to
July 20. Pythium graminicola was not cultured from roots of corn less than 14 days old or more than 132 days old. It was cultured predomi nantly during September and October. Thus, various investigators have reported that species of Pythium were readily isolated from corn roots during spring, early summer and fall but rarely during mid summer when
Fusarium spp. and other fungi were prevalent (25, 28, 82, 88).
Nematode-fungus interaction on corn: There are some indications that nematodes also may contribute to the root rot disease complex (10).
Histology of corn roots exposed to Fusarium sp and Pythium ultimum along with Ty1enchorhynchus spp. and Tylenghus spp. indicated that fungi are tho primary pathogens (42). Baldwin and Barker (4) found three species of Moloidogyne (M. aronaria, M. incognita and M. javanica) reproduced at varying rates on all cultivars of corn. They found no reproduction in corn at 25 days after inoculation, but female eggs were detected after 58 days in North Carolina. Koiki and Roman (44) in Puerto Rico found that the root lesion nematode Pratylenchus sp. caused a significant reduction in tip growth 13
of sugarcane in tho greenhouse. The root rotting fungus Pythium
graminicola significantly reduced top and root growth. When plants were
grown in the presenco of both pathogens, reduction in dry weight was the
same as those caused by tho root-rotting fungus alone. Palmer and
MacDonald (64) studied the maize root rot complex caused by Fusarium spp.
in the presence of various plant parasitic nematod species including
Moloidogyne incognita (Kofoid 6 White) Chitwood, Pratylenchus penetrans
(Cobb) Chitwood 5 Filipjev 5 Stokhoven, P. hexincisus Tayler 6 Jenkins,
£.• scribneri Steiner, Paratylenchus nanus Cobb and Tylonchorhynchus martini Fielding. Average dry root and shoot weight of maize seedlings
inoculated with both M. incognita and P., moniliforme were less than those of seedlings inoculated with either organism alone. Fusarium moniliforme decreased root and shoot weight of maize seedlings more than when tho fungus was combined with either Pratylenchus scribneri or
P. penetrans. Palmor and Kommedahl (63) found that tho dry woight of infected corn root was reduced 12% by £. moniliforme; 39% by monili- forme and Pratylenchus scribneri and 39% by £. scribneri when compared with controls. MATERIALS AND METHODS
Sampling: Tho study was conducted during tho 1974 and 1975 . growing seasons at tho OARDC, North Central Branch, Vickery and North
Western Branch, Hoytvillo, Ohio, respectively. Soil typo at the North
Central Branch was Toledo silty clay and at the North Westorn Branch was Hoytvillo clay loam. Plots sampled were part of a long term tillage-rotation experiment which is discussed in detail by Van Doren et al. (99). Tillage and rotation offects will be discussed in a separate section. Plants wero sampled by removing 25 cm^ of soil with intact root mass. Soil blocks with roots were placed in polyethylene
(1974) or paper feed (1975) bags, transported to a cold room within
2 hours, and stored overnight. Tho bulk of the soil in the block was removed from the roots by hand and replaced in the cold room until tested. This will be referred to as the soil sample. Corn roots wore washed under a cold water jet (50 psi). Isolation of Pythium species: Ten discolored root lesions from each treatment were placed on a sucrose aspargine media (SAPBNC) modi fied from Schmitthenner (69). The media consisted of: 2.5 g sucrose,
0.27 L-asparagine, 0.15 g KH2PO4 , 0.15 g K2HPO4 , 0.1 g MgS0^.7H20 ,
0.08 g CaCl2 .2li20, 0.01 g ascorbic acid, 0.002 g thiamine hydrochloride,
0.1 g neomycin sulfate, 0.005< g Chloromycetin, 0.01 g benomyl (0.02 g of 501 active Benlate), 0.02 g pentachloronitrobenzene (0.027 g of
75% active Terraclor), 0 . 0 1 g cholesterol, 20.0 g Difco agar per liter. All ingredients were added before autoclaving for 30 min at 120 C.
14 IS
Purification and identification of Pythium spp. Pythium isolates wero purified by transferring from isolation plates to sectors (1/6 of an agar plate) of selectivo medium (SAPBNC). Tho sectors were inverted in the dish to seal the lesions in a moist pocket to reduce bacterial spread. The curved, rounded edge of the sector was trimmed to ensuro that the agar surface adhered to tho glass. Species of Pythium wore identified after 2-3 days on tho isolation plate whore possible by using the criteria outlined by Middleton (55), Waterhouse (105, 106) and Mathews (49). Isolates were stored and maintained on dilute V-8 juice agar (69) slants in 15 ml bottles containing 1.0 g sucrose, 0.2 g yeast extract, 0.01 g cholesterol, 40.0 ml V-8 juice, 20.0 g agar,
960 ml distilled water. Tho V-8 juico was neutralized before adding to the media by autoclaving with 0.6 g CaCo^ and filtering through celite 545 ( John Mansvillo Co.).
Grass culture. If no distinguishing structures of Pythium isolates were formed on agar, grass leaf water cultures of isolates were studied. A small portion (ca. 0.5 cm3) of the agar was trans ferred into a potri dish containing 15 ml sterile distilled wator.Two sections of Morion blue grass, crested wheat grass or Kentucky blue grass blades, ca. 2.0-2.5 cm were boiled for 10 min placed on these • agar blocks in tho water culture and incubated at room temperature
(24 C). In another type of grass culture, sterilized ( autoclaved for
15 min at 15 psi) grass blade sections were placed on agar cultures of a test isolate and transfered after 24 hours colonization period into a petri dish containing 15 ml sterile distilled water. Plates were examined after 4-5 :days of incubation at room temperature (24 C).
For some isolates it was necessary to replace the water several times 16 or use a sporangial solution (69) to induce sporangial formation.
During this time sporangia of somo species released zoospores but
others formed zoospores after a 0.5 hours cold treatment (3 C) followed
by 0.5 hours at room temperature (24 C).
Isolation of other fungi. For isolating other fungi, 1/3 of the
same lesion used for the Pythium isolation was plated on OAES (111), a
fungus selective medium consisting of 2.0 g yeast extract, 1.0 g NaNO^,
1.0 g Kl^PO^ (mono), 1.0 g sodium propionate, 0.5 g MgS0 4 .7H20 , 5.0 g dextrose, 1.0 ox bile, 50.0 irig Streptomycin, 50.0 Chloromycetin,
20.0 g agar/liter distilled water. The remaining 1/3 of tho losion was plated on CuSO^ water agar, a Rhizoctonia-selective medium consis ting of 20.0 ug CUSO4 , 20.0 g agar/ liter wator. Ten lesions wcro plated from each trcatmont and incubated at room temperature (24 C).
Pathogenicity test. Corn kernels ( Hybrids HP 9 x OH S1A, 1975) were surface sterilized in 5% chiorox ,0 NaCIO ) for 10 minutes, washed in running tap wator for 30 minutes and. soaked .in tap wafer for
24 hours at room temperature (24 C). The soaked grains were sproad between two paper towels that were moistened and wrapped around a cylin drical bottle. The paper towel cylinders were placed in a tray contain ing just enough water to keep tho grains wet.
Petri dish water culture seedling pathogenicity tests. Five-day- old seedlings ( primary roots 3-4 cm long ) were placed in petri dishes % containing grass leaf water cultures of Pythium. Inoculum was position ed so that there was no diroct contact with the root. The cover was placed so that a 1.25 cm space was available for the leaves to grow through. No support of the plant was necessary since the roots spread
• out through tho water culture and kept the seedlings upright. 17
Test tube water culture seedling pathogenicity test. Grass culture
inoculum was prepared by placing grass blades (2.0-2,5 cm) in 10 ml of
distilled water, in a 16 x ISO mm test tube, autoclaving for IS minutes,
and inoculating with a 0.5 cm cube of SAPBNC culture of a test isolate.
After 5 days the tost tubes (cultures) were gently filled with distilled
water and the primary roots of 5-day-old corn seedlings were suspended
on nylon 'mesh in the water. Grass blade inoculum was pushed to the
bottom of the tubes with a glass rod to prevent contact between the
inoculum and root.
Both the petri dish and test tube pathogenicity tests were incuba
ted at 23 C at either 15,000 or 28,000 lux under white fluorescent tubes
and warm white fluorescent tubes plus incandescent bulbs, respectively.
Water levels were maintained by adding water whenever necessary. Root
rot was rated 5-7 days after inoculation using the following scalo:
1b small, 0.0-0.5 mm root tissue damaged; 2= large, 0 .5-2.0 mm root tissue damaged; 3° lesions coalesco to form streaks, more than 2.0-9.0 mm; 4° lesions coalesced girdling the root, 1.0-1.5 cm root rot; and
5= entiro root necrotic.
Pot culture method for screening Pythium and Fungi Imperfecti. This method consisted of mixing PDA cultures or millot seed inoculum of test
isolates with sterile soil ( two plates of PDA or 50 ml millet.seed/
1,200 ml soil ), placing the infested soil in the bottom half of four plastic, 10-cm pots, (300 ml/pot) and filling the pots with sterile soil
(200 ml/ pot) into which five corn kernels were planted. Two isolates each of tho predominant fungi isolated from corn roots were tested
( Pythium graminicola, Fusarium roscum f. sp. cerealis, £. moniliforme £. oxysporum, P. acuminatum, H . pedicillatum and Pyronochaeta terrcstris).
For millet seed culture 60 ml of seed and 40 ml of asparagine solution.
(1.13 g asparagine/liter distilled water) were autoclavcd three times for 30 minutes at 121 C at 24 hour intervals in 250 ml flasks. Seed - * was thoroughly shaken after each autoclaving to keep tho grains separate and then infected with five agar-blocks (0.5 mm^) of the test isolates.
Both agar and millet seed cultures were incubated at 22-24 C for 10 RESULTS
Symptoms of corn root rot. C o m roots examined in Juno had
scattered, yellowish-brown lesions on the first and second whorls of
secondary roots. By this time the primary root and the planted kernels
wore completely necrotic and black. As tho season progressed, the
yellowish-brown lesions coalesced and become dark brown to black resul
ting in dark streeks or girdling of roots. Frequently, fibrous roots
developed above a girdling lesion or rotted root stub. New lesions
wcro found on tho dovoloping roots that initiated tho above symptom
sequence, under conditions favorablo for root rot (Fig. 1 and 2).
In tho prosont study tho aerial symptoms such as yellowing, rolling
or wilting of the leaves, or stunting of the plant as reportod by others
(19) were not apparent. Howovor, slight stunting and some yellowing of
lower and older .leaves was obsorved in some cases early in the season.
Experimental results. To determine and recognize what fungi were
involved in the corn root rot and their prevalence during the growing
season, tho corn root samples from tillage and rotation experiments in
the field and greehouse were plated. In both 1974 and 1975, roots were
plated on SAPBNC medium throughout the growing season. The OAES medium
was used only once during September sampling of 1975 growing season
field corn roots. Tho agar sector plating method was very useful in
conserving agar and time. Secondly by putting 3 sectors separately in
a plate, the isolation and identification of fungus mycelium from a
given root tissue was easier. 19
4 20
iH
Figure 1. Corn root system with root rot symptoms. 21
Figure 2. Root rot rating, lesion typos ( s m a l l , large, streaked, girdled and necrotic). A) early in the season and D) late in the season. Lesion types were; Ismail lesions, 2° large lesions, 3=lesions coalesced forming streaks, 4= lesions coalesced and girdling root, 5«entire root necrotic. 22
Doth crested wheat grass and Merion blue grass blades were suitable
substrates for sporangial and oospore production and useful in identi
fication of Pythium species. The.grass water culture method was also
useful in producing inoculum for water culture seedling virulence tests.
Several types of Pythium spp. were recovored on SAPBNC medium.
They were 1) Pythium graminicola,identified on the basis of oospore and
lobulato sporangia; 2) a group of Pythium isolates forming lobulatc
sporangia similar to £. graminicola sporangia on isolation medium,
which will be referred to as P.G..sporangial types; 3) IP. dissotocum
and £. torulosum identified on the basis of distinctive oospore and
sporangia characteristics; 4) Pythium spp.which did not produce any
structures on isolation media but did occasionally produce lobulate
sporangin in a grass leaf culture. Because these isolates did not
produce any distinctive structures they could not be positively identi
fied; and 5) sphacrosporangial typos which did not produce oospores and
were not identified.
Pythium graminicola (types i and 2 above) was obtained in both
1974 and 1975. Pythium torulosum and £. dissotocum were isolated occasi
onally in 1974 but novor in 1975. Other Pythium spp.rwore predominantly
types 4 and 5 above in both years. Other fungi that could be detected aftor 2 weeks on SAPBNC were H. pedicillatum, Fusarium spp. and Mucor- . * ■ ales. Percentage recovery of P.graminicola, other Pythium species and other fungi is summarized in Table 1. In both tho 1974 and 1975 growing soasons, £. graminicola recovery was greatest during mid-June to mid-July and then decreased significantly with time. In June and
July platings, most graminicola were recovered from young, partially healthy roots and seminal roots. Pythium praminicola rarely was cultured 23
Tablo 1. Percentage of corn root lesions from which fungi were isolated from fiold plots at bi-weekly intervals during 1974 and 197S
Percentage of fungi isolated Percentage of fungi isolated in 1974 in 1975
Sampling b b time Pythium Other® Other Pythium Othora Othor graminicola Pythium fungi graminicola Pythium fungi
Mid-JunoC 12d 47 3 24° 16 0
Late-Junc 42 16 29 30 25 0
Mid-July I9 8 49 22 20 1 Early-Aug. 3 1 6 7 3 12
Mid-Aug. 0 0 46 1 2 41
Early-Sopt. 1 9 33
Mid-Sept. 17 14 19
LSD (.01)= 12 10 10 a Includes unidentified lobulate sporangial and sphacrosporangial types, dissotocum and £. torulosum.
^Includes Holminthosporium pedicillatum, Mucoralcs and Fusarium spp. cBi-weokly samples begining June 20, 1974 or Juno 17, 197S. tl Mean percentage of 80 lesions plated from corn roots from North Central Branch.
Moan percentage of 240 root lesions plated from corn roots from North
Central Branch. 24
from old, necrotic lesions. In late July and throughout August,
P.. graminicola recovery was the lowest in both years. In 197S, signi
ficant amounts of P. graminicola were againisolated from corn in lato
summer. Thus, a bimodal recovery curve was obtained ( Fig. 3).
Tho significant reduction in tho recovery of pythiaccous fungi
during mid-summer was associated with low rainfall (Table 2). In late
August and early September (after tasscling) following a rainy period,
Pythium species were again recovered in moderato frequency. In 1974,
H. pedicillatum isolations followed the same pattern as the P. grami
nicola but fewer were obtained. Other fungi were recovered most
frequently from mid-July sampling onwards (Table 1, Fig. 3). Predomi
nance of all othor fungi found associated with c o m root rot during
the late season were investigated by plating on OAES medium.
Of 240 lesions, Pyrenochaeta torrostris (22%) and Fusarium spp.
(38%) were isolated in greatest numbers. Trichodorma spp. (14%)
appeared on many plates whon incubated for moro than 2 weeks. Helmintho-
sporium sp. (6%), Penicillium sp. (6%), Mucorales, and Cladosporium sp.
(9%) wore found infrequently in some platings. Fusarium spp. found
wore: F_. rosoum, £. moniliformo, J?. oxysporum, F, acuminatum, F. solani
and £. cquisoti.
A final sampling of corn roots was made from corn growing in soil
from the North Western Branch in the greenhouse. Samplings were at
I, 2, and 3 months after planting. In addition to SAPBNC and OAES a
CuSO^ medium was used to‘see if Rhizoctonia was also involved in the root rot complex. Pythium graminicola recovery was high at 1 month, less at
2 monthsand least at 3 months. Fungi recovered in the greenhouse are presented in Tablo 3. Recovery of other Pythium sp. (mostly lobulate Table 2. Bi-weekly summer prccipitaion at the North Central Branchy 1974* and the North Western Branchy 1975, OARDC
Bi-weekly Total precipitation period 1974 1975 a June 1-Juno 15 2.20 2.35
June 16-June 30 2.54 1.24
20 year average 3.74 3.69
July 1-July 15 0.69 1.43
July 16-July 31 0.31 0.36
20 year: average 4.57 4.03
August 1-Aug. 15 1.95 4.01
August 16-Aug.31 2.09 5.11
20 year average 3.42 2.99
Sopt. 1-Sept. 15 0.54 2.87
aInches of rainfall. Percentage fungus recovery 30 Fugure 3. Percentage of corn root lesions from which fungi were fungi from lesionswhich corn root Percentage of 3.Fugure June recovered in197S,recovered June uy July July 4 28 14 Sampling time Sampling Other Pythium spp. LSD (.01)«10.41 LSD spp. Pythium Other Other fungi Other yhu rmncl LD (.01)=12.5 LSD Pythium graminicola u Aug. Aug S (.01)=9.94 LSD Sept 26 27
Table 3. Pungl recovered on SAPBNC media at monthly intervals from roots of corn growing in the greenhouse in fiold infested soil
Percentage of lesions with fungi
Sampling Pythium Other Other time graminicola Pythium fungi
1-month 60a 50 10
2-month 40 30 30
3-month 20 40 80 .
a Based on 120 lesions plated/sample. 28 sporangial typos) decreased with time but not significantly, Recovery
of other fungi on SAPBNC was rare during the first sampling, but tho numbers increased from second to third sampling periods. On OAES medium
Fusarium spp. were recovered in great numbers, particularly during tho
second and third sampling. A few Pyrenochaeta wero. isolated as the
season progressed. Holminthosnorium spp. recovery was much higher on ,
SAPBNC than on OAES medium. A few spherical sporangial pythia were recovered at all sampling times and wero included among other fungi.
No Rhizoctonia was isolated. Pathogenicity test results. Root development was bettor and results were more consistently reproduced using potri dishos rather than test tubos in the water culture socdling pathogenicity tests. However, in the test tubes a largo number of isolates can be scrocncd in a limited space. In both methods, tho seedlings were infected and ready to exami ne after 5-6 days of incubation at room tomperature <24 C) or at 23. C in a growth chamber. Results of tho petri plate water culturo seedling pathogenicity tests are summarized in Table 4.
Corn seodlings showed various degrees of symptom expression.
Small yellowish-brown root lesions were visible within 4-5 days after exposing to 5-day-old water-grass culture inoculum. In severe casos the seminal roots were also boen attacked. Usually, roots with lesions on one side would bend towards that side. Sporangia and oospores were observed in infected roots when cultured for 2-3 woeksv Aerial port-
* ions of the plants usually appeared normal 5-6 days after examination.
However, in somo cases whore primary roots were severely damaged, the leaves were either twisted, curled or showed tip burn. 29
Table 4. Relative virulence of Pythium spp. on corn seedlings using the petri dish water culture seedling test
Fungi Root rot rating
1. Pythium graminicola 4a (oospore typo)
2. P. graminicola 4-5 (sporangial typo)
3. P. dissotocum i-2
4. P. torulosum 1-2
5. P. graminicola 5 6 P. torulosum
Based on three tests of 20 isolates, oospore type and 6 isolatos of sporangial typo of Pythium graminicola and 6 isolatos each of P.. dissotocum and £. torulosum whore 1® small, 0.0-0.5 mm root tissuo damaged; 2° largeT 0.6-2.0 mm root tissuo damaged; 3« lesions coal esce to form stroaks more than 2.0-9.0 mm; 4“ lesions coalesce girdling tho root; 1.0-1.5 cm root rot; and 5« cntiro root necrotic. 30
A detailed study of the infection process was made with an isolate of P. graminicola in a corn seedling water culture. Within 24 hours of introducing the seedlings, zoospore clumping on tho root surface was observed. Zoospores encysted on the root surface and germinated by producing germ tubes; but in some instances the mycelium itself was capable of ponetrating and infecting. The fungus developed inter-and intracellular mycelium and colonized tho cortical region of the root.
The root elongation zone and tertiary roots were very susceptible.
Infected roots may or may not rot completely. Root pruning was obser ved when progressive types of lesions developed. This was a common phenomenon obsorved with £. graminicola isolates. Pythium dissotocum and P. torulosum caused a non-progressive type of lesion.
Biflagellate zoospores were observed in 5 to 6-day-old grass water culture dishes. The cytoplasmic contents from lobulate sporangia in
graminicola and P_. torulosum or filamentous sporangia in P.dissotocum empty into a thin walled vesicle through a narrow emission tube. The zoospores cleaved and escaped from the vesicle and swam away by a glid- ing motion. Spores came to momentary rest and encysted and germinated within an hour and repeated the asexual cycle. £. graminicola zoospores could be readily distinguished from P. torulosum and P. dissotocum by their large size (10 u vs. 7 u), The established life cycles of theso..
Pythium spp. occurred in the water culture seedling plates.
Soil seedling pathogenicity test. This test studied the virulence of different fungi isolated from corn roots and also determined the interaction of j>. graminicola with, other fungi. No root rot was obtai ned with any of the fungi tested using agar cultures as inoculum.
Thirteen days after planting in artificially-infested sterile soil with 31 millet seed inoculum the seedling emergence, root rot ratings, root and top length and dry weights were taken. Differences in plant height, leaf color and root growth wore evident when plants were in 3 to 4-leaf stage. No significant differences in emergence wore evident.
In all treatments, tho primary root was necrotic. General examin ation of infected roots indicated that more roots had lesions or were rotted C*oot pruning) by P. graminicola than by the other fungi tested.
Pythium graminicola in combination with P.. oxysporum, £. moniliformo,
H. pedicillatum or P. terrostris caused significantly moro root rot than
P_. graminicola alone or in combinations with F_. roseum of F. acuminatum.
Helminthosporium pedicillatum caused a few scattered lesions and tho root mass was not reduced significantly. Fusarium roseum. JP. monilifo- rmc, and JP. oxysporum reduced root mass and top growth. A few scattered and necrotic lesions wore incited by £. roseum and oxysporum. Even though the root mass was reduced by JP. moniliformo. few lesions were prosent and roots appeared white, clean and fibrous. Fusarium acumina tum did not produco symptoms or reduce tho root mass compared to the control. Pyrenochaeta torrostris caused rcddoning of tho roots with abundant scattorcd lesions, but root mass and top growth were only slightly reduced. Stunting of top growth and leaf tip burn wero most evident with P.. graminicola plus P. terrestris. F. oxysporum or
F. moniliformo. DISCUSSION
From tho rosults obtained in this investigation I have concluded fchat Pythium graminicola is tho initial incitant of corn root lesions and root rot early in tho season. Other fungi colonize the root tissue as secondary invaders and increase root decay during mid and late season.
However, P. graminicola appears again during late summer as tho plant matures.
Tho extensive root damage evident in late August onwards could not bo explained completely on the basis of P. graminicola. It was tho only fungus tested that was highly virulent on corn seedlings (S days-3 weote). However, none of the fungi isolated were tested on older corn. The exact etiology of late season root rot was not determined in this invest- gation. Pythium graminicola and H. pedicillatum were the predominant fungi rocovered from root lesions early in the season. In mid season pythiaceous fungi decreased or wore absent, even though necrotic roots or those with necrotic lesions were present. Spocies of Fusarium and
Holminthosporium wore recovored from such necrotic roots. In late summmer following a Wet' period P. graminicola was isolated from lesions - on, fibrous roots developing from prop roots. However, it was less preva lent than H. pedicillatum, and Fusarium spp. Therefore, in the last sampling, roots were plated on OAES as well as SAPBNC to determine all fungi associated with necrotic roots. A large number of imperfect fungi,
Fusarium roseum, £. monilifonne, £. acuminatum, F. oxysporum, H . pedi cillatum and P. terrestris were recovered. Data presented here on the 32 33 recovery of P. graminicola followed tho same pattern in both 1974 and
1975 and was least in late July and August. Moisture and temperature are reported to be responsible for the decrease in tho P. graminicola and other pythiaccous fungi during mid season by Hampton and Buchholtz
(25). They reported that tho frequency and occurrence of different groups of fungi from corn root rot is positively correlated tvith the precipitation data and negatively correlated with average soil tempera ture . The lowest Pythium rocovery occurred in the present study in 1975 during tho period of low rainfall in July. Pythium was again recovered in September about 1 month after the onset of a wet period.
Since a general fungus medium, OAES, also was used in the green house tests, it was possible to follow the prevalence of non pythiace- ous fungi,(specios of Fusarium. Holminthosporium and Pyronochaota ).
Surprisingly, £. graminicola was the most prevalent fungus isolated from corn root lesions at the first sampling time (1-month-old plants).
Apparently, £. graminicola is tho only significant pathogen on young plants. Othor fungi were moro prevalent at the second sampling period and most prevalent at tho third sampling period (3-month-old plants).
On tho other hand, the frequency of £. graminicola decreased as the plant matured evon though soil moisture was maintained close to satu ration. Based on prevalence of fungi alone, it would appear that early * « root damago was caused by Pythium and late root damage caused by a complex of species, Risarium, P. terrostris and H. pedicillatum.
This data may indicate that the maturity of corn may bo responsible for the decrease in Pythium rather than moisture and temperature as suggested by others. More experimentation is needed before concluding the relative importance of maturity and moisture on tho prevalence of 34 Pythium root rot of corn.
However, the non-pythiaccous fungi isolates were not virulent on
young corn. In contrast to the work of Shephard ot al.(74) .onH.'pedi-
fci.llatum, very few root lesions and no discoloration of the roots deve
loped in my trials. It is possible that more damage could have been
obtained with other isolates of H. pedicillatum or other hybrids or more mature plants. More work needs to bo done before H. pedicillatum
can be excluded as a corn root rot pathogen.
None of the 'Fusarium spp„ caused significant root rot in pathogeni
city tests, although F.. moniliforme and £. oxysporum did significantly reduce root mass. This root inhibition could be the result of toxic metabolic products accumulated in tho millet socd inoculum. Many others have reported that Fusarium spp.are generally secondary invaders and weak parasites (28) which agrees with the results obtained in my studies.
The virulence of Fusarium on older corn needs to be tested before exclu ding members of this genus as corn root pathogens.
The only other potontial pathogen obtained was Pyrenochaeta torre-* stris that causes a red root rot of corn (8, 21, 39). Rod root rot was observed in 197S late in the growing season and at the third sampling
(3-months-old) in greenhouse tests. Pyrenochaeta terrestris was isolated from such roots occasionally. It produced a large number of reddish scattered lesions but did not reduce root mass and top growth, which agrees with the work of Craig and Koehler (13).
Tho cause of severe root rot observed late in the season is still undetermined. A number of possibilities can be postulated:
1 . Pytliium graminicola can initiate lesions that are rapidly taken over by secondary fungi and may not persist in lesions colonized by other fungi. 35
2. Corn plants lose resistance to weak pathogens such as Fusrium spp.
H. pedicillatum, £. terrestris as they mature,
3. Various combinations of weak pathgens may act syncrgistically to
cause corn root rot.
4. Other components of soil fauna, i.e. nematode and insects, may interact
with weak pathogens to produce lesions.
5. Roots may become so senoscent that they are rotted by saprophytic
microflora, i.e. species of Trichoderma, Mucoralcs, PeniciIlium and
Aspergillus.
6. Other fungi not isolated in this study might be involved,for example
McKeen 151) has roportiJd Phialophora radicicola to b6 highly virulent
on corn^but requires special.techniques for iits.isloations.
Two Pythium species torulosum and £. dissotocum, were isolated infrequently from field infected roots. The water culture pathogenicity test indicated that they wero weak pathogens. Probably, they are not imortant in corn root rot. Considerable effort was spent with green house tests to isolate Rhizoctonia but nono wero obtained. Ho (28) indicated that Rhizoctonia can infect roots and roduce top growth in corn. However, Rhizoctonia should have been isolated if it had been involvod in the c o m root disease complex.
Media used in most of this investigation, SAPBNC was supposedly selective for Pythium spp. only. However, it also was found to be very selective for H. pedicillatum and more of this fungus was obtained on
SAPBNC than OAES used in the latter part of the study. OAES was quite satisfactory for routine isolation of Fusarium and Pyrenochaeta and other common soil fungi. PART II
PYTHIUM ROOT ROT OF CORN: METHODS FOR DETECTION OF
PYTHIUM GRAMINICOLA IN SOIL
36 INTRODUCTION
Root rot may bo implicated in the lower than expected yields of corn in certain crop sequence and tillage systems (14). In Ohio on poorly.drained soils, corn yields wero reduced 10r20% under a continu ous c o m nontillage system as compared to corn in rotation but with conventional tillage on tho same soil type. Tho cause of this yield reduction was not established (91, 99). However, it was observed (99,
Schmitthenner unpublished) that the root system was less extensive in nontillcd, continuous corn than in tilled, rotated corn (99). Williams and Schmitthenner (109) found that root rot was worso in continuous corn than in a corn-soyboan rotation scquonce. They also established that corn yields wore negatively correlated with corn root rot. In their study, root rot of corn was loast and yields highest when corn followed soybeans while severe root rot and lowest yields occurred in continuous corn. However, tho cause of the root rot was not established.
Johann et al. (41) reported that Pythium caused a 31% reduction in aver age seedling stand of corn and a 65% reduction in yields in an Illinois test.
In ray study, it was observed (Part I) that continuously cropped nontillod corn was slightly stunted and light green. Infected roots were rotted. Infection usually occurred at tho tips of rootlets and proceded proximally, producing a soft rot, involving first the cortex
37 and later the vascular elements. It was concluded that Pythium
graminicola Subr. is the initial incitant of corn root lesions and root
rot during tho early season and that other fungi colonize the root
tissue as secondary invaders and increase root decay during mid and
late season (Part I).
It is possible that rotation and tillage could affect root rot by
influencing a) the numbers and types of root rot pathogens, b) tho
germinability and virulenco of pathogens, c) repressions of pathogens
by associated soil micro-organisms and d) the suscoptibility of roots
to infection and colonization.
Many investigators havo reported the occurrence and association of different organisms with root rot of corn and their virulenco. Prom . previous work it is apparent that £. graminicola was the primary patho gen of corn root rot in heavy soil (Part I).
To determine tho effect of tillage and rotation on £. graminicola, it is necessary to evaluate its relative abundance in soil. So far, attempts to isolate j\ graminicola directly from soil have been unsucc essful rogardloss of the method used. Many investigators have contri* buted to the development of effective and selective isolation techniques for several species of Pythium. Detailed information can be found in
Eckert and Tsao (17), Mircetich. and Kraft (56), Singh and Mitchell (78),
Tsao (93), Schmitthenner (70) and Hendrix and Campbell (26). These methods are suitable for quantitative estimation of some Pythium spp. ■ Methods triod without success for isolation of P_. graminicola were; a) screened immersion plate method (112), b) soil particle method (70), and c) serial dilution end point method (94), 39
Baiting techniques have been used successfully for isolation of
£.• graminicola. Buchfioltz used a crested whcatgrass (Agropyron cresta-
ta) seedling bioassay (7). Pythium graminicola is a principal cause of
seedling blight of crested wheatgrass. Seedling root trap methods were
used successfully for Phytophthora (1, 9, 48) which is also very diffi cult to isolate directly from soil. In this investigation a crested
whcatgrass seedling assay and a corn seedling root bioassay are descri
bed that are useful for detection of P_. graminicola in soil. Using
these methods it was possible to demonstrate the activity of £. gramini
cola in mid'summer when P.. graminicola could not be isolated from corn
root lesions. MATERIALS AND METHODS
The soil for crostcd whcatgrass seedling bioassay tests (CWG) and tho corn seedling root trap tests (CRT) was obtained from continuous corn in a long-term tillage experiment at the North Central Branch,
OARDC, Vickery, Ohio, site 4, as described by Van Doren et al. (99).
The soil type was Toledo silty clay. Tho soil was sampled five times by removing ten corn plants with attached ca. 25 cm3 soil mass from a conventionally-tilled plot. Tho plants with soil were transported to
Wooster, placed in a cold room overnight, and the soil was mechanically removed from the roots the following day.. Soil from each corn plant was stored separately in a cold room until use.
Crested wheatgrass seedling bioassay (CWG): For tho CWG bioassay eight 200 ml aliquots of soil from each c o m plant wore placed in indi vidual styrofoam cups. Four were planted with 50 seeds of cultivar
NorIon and four with cultivar Intermedia and covered with ca. 25 ml vermiculito. The cups were placed in greenhouse and watered daily. For the control, soil from both tilled and nontillcd plots was steamed and four styrofoam cups wero used for each soil-CWG variety combination.
One and 2 weeks after planting the total number of healthy seedlings
* was recorded. Two diseased seedlings were selected at random from each cup, the roots were washed in tap water and then distilled water and plated on selective media to verify the presence of IP. graminicola.
40 41 Data were expressed as numberof healthy seedlings at the end of a
2-week, period.
Corn seedling root bioassay (CRT]: All the subsamples from till
ed and nontilled plots were bulked by plots after removal of 200 ml
aliquots for CWG assay. From this soil mixture 100, 200 and 300 ml
soil samples wore put into 500 ml flasks, with four replications of each. Those flasks wore marked appropriately and gently filled with
tap water without disturbing the soil, then allowed to settle so that any floating materials could accumulate at the neck of the flask. After
6-8 hours of flooding, the floating materials were removed. Five-day- old corn seedlings obtained by the paper towel germination method, desc ribed in Part I, were suspended in water over the soil with nylon mesh.
Those flasks were thon placed under a sprinkler in the greenhouse
(temperature ca. 28 C) for 5 days with 5 minute sprinkling cycles of deionized water per day.
After fivo days of incubation, corn seedlings from each flask were removed, thoroughly washed under running tap water, and their primary roots were placed in water in a.potri dish for examination. Discolored, brownish-yellow lesions were counted using a dissecting microscopo and classified into the following types: 1= small, 0.0-0.5 mm root tissue damaged; 2° large, 0.6-2.0 mm root tissue damaged; 3= lesions coalesce to form streaks more than 2.0-9.0 mm; 4* lesions coalesced to girdle the root, 1.0-1.5 cm root rot; and S« entire root necrotic. A total of
48 lesions (2 lesions/flask x 4 replicates x 3 soil volumes x 2 tillage treatments) were plated on a Pythium selective medium (SAPBNC) and plates were-examined for Pythium after 4 days of incubation at room temperature (24 C) as described in Part I. 42 Numbers of each type were expressed as average number of lesions per samplei and the average number of lesions types 1 and 2 and 3, 4, and S, respectively, were combined. Avorage lesion score consisted of the sum of number of lesions of each typo x the lesion type divided by total number of lesions. For statistical anlysis, the four subsamples of each replication were combined. A factorial analysis of variance was computed for each set of data. The least significant difference was used for mean comparisions. RESULTS
Crested whoatgrass seedling bioassay (CWG); Infected plants were stunted, with chlorotic leaves. Eventually, some plants wilted and the leaves dried. In somo instances, seeds did not germinate and in other cases post-emergence seedling blight resulted in stunting and death of seedlings(Fig. 4). One week after planting there was more post-cmergcnco damping off of Norlon than in Intermedia cultivar and in field soil than in steamed soil. Post-emergence damping off showed no consistent trend in soil sampled at different,times during the summer. Differences woro much more striking 2 weeks aftor planting and these data wore used for evaluating the tcchniquo. -At 2 weeks, significantly more post*emergcnco damping off occurred in Norlon than in Intermedia. Pythium graminicola was obtainod consistently from infected seedlings of both varieties. Damping off varied significantly with sampling time, but no seasonal trends were evident (Table 5).
Power seedlings survived in tilled (6.8) than in nontilled soil (15.4).
Interactions of sampling time, crested whoatgrass cultivar, and soil types sampled were significant (Table 5). It is apparent that
Norlon is more sensitive to damping off by £. graminicola than Interme dia. It is also evident that least damping off (29-41 surviving plants) occurred in steamed soil and most in tilled soil ( d-14) surviving plants) at four out of the five sampling times. Damping off was inter mediate in the nontilled soil (6*5-17 surviving plants). In general,
43 Figure 4r(A to B). Crested whoatgrass seedling bioassay (CWG) method to detect levels of Pythium' spp. in infested soil (tilled and nontilled soil). Treatments are cultivar Norlon in A) autoclaved, B) tilled, and C) nontilled soil* and cultivar Intermedia in 0) autoelaved, B) tilled, and F) nontilled soil 4S
Tabic S. Number of surviving seedlings of two varieties of crested whoatgrass. two weeks after planting in soil from tillage plots
Crested wheatgrass Cultivar •
Intermedia Norlon
Sampling Stcamoda Tilled Nontillcd Steamed Tilled Nontilled time (1974) soil soil soil soil soil soil
July S 38b 12 21 29 14 17 July 19 45 5 12 37 0.25 8
Aug. 2 44 11 26 38 0 7
Aug. 16 38 12 15 31 0 11
Sept. 3 44 10 24 41 4 14 LSD (.05)“ 6.5 a Mixture of tilled and nontilled soil. b Mean number of surviving seedlings out of four pots of 50 seeds each, two weeks after planting. 46 damping off was moro severe in Norlon in the tilled soil at the last
four sampling dates as compared to the first sampling date. Differen
ces were not as great with Intermedia. It is concluded that Norlon is
more suitable than Intermedia as a seedling bioassay indicator plant.
Also, it is possiblo to detect differences in level of P. graminicola
in different tillage systems, and therefore the CWG assay might be
suitable for determining effects of other cultural variables, i.e.
rotation, drainage etc.
Corn seedling root bioassay (CRT); Minute yellowish-brown lesior-
ns wore visible 3 days after suspending seedling roots in water over
soil. At the end of 5 days all five types of lesions could be recogni
zed as described before. Roots were stunted and curved at regions whore
many small lesion types or lesion streaks were present. Lesion typos
1 and 2 were most abundant. Generally only one streaking (type 3),
girdling (type 4) or necrotic (typo 5) lesion was present on a primary
root. In sovore cases, the coleoptilo failed to emerge and secondary
seminal roots also were attacked. Results of the CRT assay are summer-
ized in Tables 6-8 , Pig. 5. Different quantities of infested soil
samples were used to determine the amount which wduld give the least variable results.
Average number of lesions and average number of small lesions
Cl and 2 type) did not differ significantly with sampling time when
200 or 300 ml of soil was used (Table 6). Average lesion score varied with sampling time irrespective of the amount of soil used for the assay (Table 7). There was no trend in lesion score throughout the season. Average number of large lesions decreased from July-September sampling times using 100 or 200 ml soil but did not differ significantly 47
m *
B
Figure 5-(A,B). Corn seedling root trap bioassay (CRT) method to detect levels of Pythium spp. in infested soil: A) 5 days; B) 2 weeks after suspending corn seedlings in water over soil. T® tilled soil, NT* nontilled soil, C® control, steamed soil. 48
Table 6. Mean number of total lesions and small lesions on corn seedlings roots suspended* in water over three different volumes of soil (CRT bioassay) collected at five sampling dates
Number of lesions
a Sampling Total lesions/ root using Small lesions/ root time soil volumes of using soil volumes of
100 ml 200 ml 300 ml 100 ml 200 ml 300 ml
b July 5 13.6 13.6 8.9 4.8 4.6 2.6
July 19 11.6 10.5 10.3 3.8 3.5 3.4
Aug. 2 7.0 9.8 9.6 1.8 3.3 3.6
Aug. 16 10.1 11.5 10.8 4.3 4.9 3.8
Sept. 3 8.0 8.6 12.8 3.1 3.2 4.4
LSD (.05)= 3.8 NS NS 2.1 NS NS a All lesions 2 mm diameter or smallor. b Mean number of lefiions/ plant from four plants from four assay units for 10 soil samples from two tillage plots (320 roots). 49 Table 7. Mean lesion score and number of large lesions on corn . seedling roots suspended in water over three different volumes of soil (CRT bioassay) collected at five sampling dates'
Variables measured
Mean lesion score Mean number 6f large using soil volumes of lesions using soil volumes of
Sampling 100 ml 200 ml 300 ml 100 ml 200 ml 300 1 time
July 5 2.2° 2.1 2.3 1.3 1.5 1.2
July 19 2.0 2.1 2.0 1.0 1.2 1.1
Aug. 2 2.4 2.0 1.8 1.1 1.0 0.8
Aug. 16 1.6 1.7 2.0 0.5 0.6 1.0
Sept. 3 1.7 2.0 2.3 0.6 0.75 1.3
LSD (.01)= 0.38 0.2. 0.55 0.61 NS a Numbor of losions of each type x the losion type devidcd by the number of lesions where lesion types were: 1° small, 0.0-0.S mm root tissue damaged; 2= large, 0,6-2.0 mm root tissue damaged; 3= lesions coalesce to form streaks more than 2.0-9.0 mm; 4° lesions coalesced to girdle the root, 1.0-1.5 cm root rot; and 5= entire root necrotic. b Lesions 2 mra or greater. c Based on four plants from four assay units for ten soil samples from two tillage plots (320 roots). 50 using 300 ml soil (Table 7). Tillage differences could be detected using 200 or 300 ml soil for the assay and the lesion score small lesion, or largo lesion variable
(Table: 8). Lesion score and large lesion number were higher in tilled than nontilled, but the number of small lesions higher in nontilled than tilled soil. Pythium graminicola, torulosum, £. dissotocum were isolated using this seedling bioassay method. Pythium graminicola was recovered predominantly from large type (types 3, 4, and 5) while all three species were isolated from small lesions (types 1 and
2). It was concluded that both the CWG and CRT methods were suitable for detecting P. graminicola . in soil and useful for evaluating telat- ive levels in soil.. 51 Tabic 8 . Mean lesion number, lesion score, small lesions and large lesions on corn'seedling roots suspended in water over three different volumes of soil ( CRT bioassay) collected from two tillage systems on Toledo silty clay soil
Variables measured
. a b c Soil Tillage Total Lesion Small Large volume system lesions score lesions lesions
100 ml Till 9.3d 1.9 3.3 0.9
No-till 10.3 2.0 3.7 0.9
LSD (.01) NS NSNS NS
200 ml Till 9.0 2.1 3.0 1.2
No-till 12.0 1.8 4.7 0.8
LSD (.01) NS 0.17 1.6 0.39
300 ml Till 10.0 2.2 3.1 1.3
No-till 10.9 1.9 4.0 1.0
LSD (.01) NS 0.24 0.8 0.25
a Number of lesions of each type x the lesion type dovided by the number of lesions where lesion types were: 1= small, 0 .0-0 .5 mm root tissue damaged; 2= large, 0 .6-2.0 mm root tissue damaged; 3° lesions coalesce to form streaks more than 2.0-9.0 mm; 4= lesions coalesced to girdle the root, 1.0-1.5 cm root rot; and 5° entire root necrotic. b Lesions 2 mm or smaller. c Lesions 2 mm or greater. d Based on four plants from four assay units for ten samples from two tillage plots (320 roots). DISCUSSION
In Ohio, Pythium graminicola is the primary causo of root rot of.
corn on certain poorly drained soils. Other imperfect fungi increase the
root damage as secondary invaders or by colonizing dead roots.(Part I).
In general Pythium spp. are widely distributed and most can be easily
isolated by the conventional soil or root plating techniques. However,
jP. graminicola has never been isolated directly from soil although it
has been isolated readily from infected roots. This pathogen has been
recovered from diseased corn, roots early in the season or from maturing
plants but not in mid-summer when the root damago was oxtensive (Part 1}.
To determine if this decline in root damage and absence of £. gramini-
cola in root was due to a decrease in inoculum level in soil, it was
necessary to dcvolop techniques to estimate £. graminicola in soil.
One method tried was the crested whoatgrass seedling bioassay,
first used by Buchholtz (7). In my studies graminicola proved to bo
the primary pathogen, causing post emergencedamping off and seedling
blight in crosted whoatgrass, as reported by Buchholtz (7) and Andrews
(2). Norlon variety was more susceptible than Intermedia and therefore
is a more sensitive indicator plant. Therefore, Norlon cultivar would be more suitable for estimating graminicola under conditions (dry, high temperature) where levels of the pathogen would be anticipated
to be low. This method is quite simple and fast and is useful in - screening a large number of infested soils for P. graminicola.
52 53
The second method successfully used for P_. graminicola from soil was a c o m seedling root trap method. Using this method £. graminicola,
£.• torulosum, and P. dissotocum were the predominant species isolated from corn roots in my study. Pythium graminicola was found associated with progressive lesions (typos 3, 4, and S) while £. torulosum and and. P. dissotocum were isolated only from small lesions (typos 1 and 2)..
Several Pythium spp. which did not sporulato readily were also obtained.
Those proved to be mostly P.. graminicola and occasionally torulosum when eventually identified from structures formed in grass water culture.
Usefulness of these two methods for detecting Pythium graminicola in soil can be summarized as follows:
1. P. graminicola could be demonstrated in soil throughout tho summer
oven during the periods when it was not isolated from c o m roots.
2. There were no consistent seasonal trends in tho amount of ]?. grami
nicola dotectcd using tho CWG mothod (Table. 5). No seasonal trends
were noted with the CRT method using average number of lesions,
average number of small lesion, or average lesion score variables.
However, a significant decrease level of P. graminicola with time
was indicated by a decrease in number of largo lesions, when 100 or
200 ml of’soil was used. 3. More P.. graminicola was found in tilled soil by.the CWG method, or
the CRT method using lesion score or number of large lesions than
in nontillcd soil.
4. More small lesions were found on roots using the CRT method in non
tilled than in tilled soil-probably because small lesions were
caused by £. torulosum, P_. dissotocum and I\ graminicola.
5. In conclusion, the CWG mothod is simpler and more consistent but 54 less sensitive than the CRT method. It*was useful for detecting
tillage and seasonal differences.
6 . The CRT method appeared to be most consistent using 200 ml of soil
and numbers of large lesions for measuring treatment differences.
7. In general, total lesion counts and the number of small lesions were
least consistent with, the CRT method. Losion score was better but-,
less sensitive than the large lesion counts.
8 . The number of large lesions was useful for detecting both tillage
differences and seasonal differences. The lesion score was suitable
for detecting tillage differences only. The total number of lesions
or number of small lesions were least useful for evaluating
graminicola .
9. Both CWG and CRT methods should bo suitable for detecting P. gramini
cola in a wide variety of cultural treatments.
10.The CRT .Method could be adapted for screening genetic lines for
resistance to natural population of £. graminicola in the soil.
11. Both mothods could be useful in evaluating the relative abundance
of £. graminicola in infested soil. Tho CRT method using the right
amount of soil and counting the progressive lesions should provide
tho best available method for estimating £. graminicola levels.
12. Slightly virulent torulosum, and £. dissotocum, as well as
highly virulent P. graminicola, were obtained using this method. In
some lesions, both P. graminicola and £. torulosum wore present.
However, P. graminicola could be distinguished because it was the
only species which, produced progressive lesions. PART III
PYTHIUM ROOT ROT OF CORN: EFFECTS OF TILLAGE, ROTATION,
FUNGICIDES, MOISTURE AND TEMPERATURE.
SS INTRODUCTION
Even though root rot of corn has been implicated in the poor yields
in certain crop sequences and tillage systems (14)* the disease has not
been studied to any extent. Dickson (14) discussed corn root rot
caused by P.. graminicola and IP. arrhenomanes. However, the corn root
rot was not mentioned in the recently published Compendium of Corn
Disoases (77), although it did discuss c o m seedling blights caused
by Pythium spp. other than £. graminicola. Root necrosis and root
rot of corn is a woll established disease (Part I).
In Ohio on poorly drained soils, corn yields vrere reduced 13%
in nontilled corn as compared to corn in rotation and conventional
tillage (14). Similar yield loss of corn in nontillcd poorly-
drained soils have been reported elsewhere (22, 23). The cause of this yield depression has been suggested to be P. graminicola (Schmitthennor- personal communication). It has been established that corn yields were negatively correlated with corn root rot. Best yields and least root rot were obtained following soybeans and lowest yields and most
severe root rot was present in continuous corn (111). However, the cause of the root rot was not established. Richardson (66) reported most Pythium (mostly P. graminicola) root infection of corn following timothy and least following soybeans while in a corn-corn sequence the
disease incidence was intermediate. Staffoldt (83) found less corn root necrosis caused by £. graminicola in a corn-oat-meadow rotation
56 57 than in corn-oat-meadow-meadow, corn-corn~oat-meadow or corn-oat rotations.
Information on the offcct of temperature and moisture on the incidence of root rot of corn is limited. It has been estimated that
Pythium root rot in general is favored by high soil moisture and moderatoly low soil temperatures. Johann et al. (40) reported that post emergence damping off of corn by Pythium was enhanced by low tcmporature and high lovols of soil moisturo. Low soil temperature with soil moisture at 70% or more of saturation were reported by Ho
(28) to be most favorable for tho infection of corn seedling by P. debaryanum. McLaughlin (52) found that Pythium was readily isolated in cool and wet soils but rarely when soil temperature was high and soil moisture low. Other workors have shown that Pythium causes root necrosis of seedling corn, especially at soil temperatures of 22-27 C.
(24, 28, 34, 37). Pythium root rots of other crops arc also affected by temperature and moisture. Flor (20) demonstrated that Pythium damage to sugarcane was groatest at moisture levels above 50% of soil water-holding capacity. Ho also found that Pythium reduced germination and subsequent growth of sugarcane most at low soil temperatures. At
35 C, Pythium did not injure seedlings while at 30 C there was an appreciable amount of injury and as the temperature was lowered injury was even more severe. Vanterpool and Truscott (101) observed that the damage to wheat seedlings by a Pythium sp. increased with increasing moisture content of the infested soil.
Various investigators have reported that species of Pythium were readily isolated from soils during the spring and fall but rarely during the hot summer (.25, 28, Part I). Because P. graminicola is the primary roct pathogen of corn in some soils in Ohio (Part I), it
seemed desirable to investigate some of the environmental factors
influencing the survival of Pythium in soil. The effect of soil
temperature and moisture seemed especially pertinent and was evaluated
in the greenhouse and growth chambers. Since £. graminicola was the primary corn root rot pathogen in situations already reported (Part I
5 II) it seemed desirable to further establish its contribution to root rot and yield reduction by controlling'it With chemicalsor other means..
Two chemicals have recently been used to control Pythium on other crops, ethazol CTruban) and Pyroxycholor (Dowco 269) (34). Neither of these 4 has been used for field crops extensively and never on com, because they are best suited for application as drenches.
In Ohio, £. graminicola initiates root lesions early in the season and Fusarium oxysporum, F. moniliforme, roseum and F_. acuminatum,
Pyronocheata terrestris and Helminthosporium pedicillatum colonize these lesions as secondary invaders and increase the root rot, often resulting in a necrotic black root system by late in the season. Only
P. graminicola was prevalent on young roots and it was highly virulent on corn seedlings (Part I).
Using corn and crested wheat grass (Agropyron crestata var. Norlon and Intermedia) seedling bioassays, it was possible to determine the relative abundance of JP. graminicola in infested soil in a long-term tillage experiment (Part II). In the present study the effects of tillage, rotation, and fungicides on root rot of corn and levels of
I\ graminicola in soil were investigated using tillage-rotation plots at tho North Western Branch, OARDC, Hoytville, Ohio. Effects of temperature and moisture on c o m root rot were investigated in growth chambers and the greenhouse using soil from tho tillage-rotation plots
Control of root rot was attempted using Pythium-specific fungicides-
Pyroxycholor (Dowco 260) in growth chambers and greenhouso and Truban in the greenhouse. MATERIALS AND METHODS
Experimental design. This study was conducted in 1974 at the
North Central Branch, OARDC, Vickery, Ohio and in 1975 at the North
Western Branch, OARDC, Hoytville, Ohio, sites 4 and 3, respectively, described by VanDorcn et al. (99). At both locations, three rotation variables and three tillage variables were established in a complete factorial randomized-block design with three replications. At the North
Central Branch the two treatments sampled wero continuous corn non- til lago and continuous corn conventional tillage. At the North Westorn
Branch the four treatments sampled were: 1) continuous c o m notillage,2) continuous corn conventional tillage, 3) corn-soybean, no tillago, and
4) corn-soybeans conventional tillage. The conventional tillage plots wore plowed in fall or early winter. Fine, firm seed beds were prepared with disk and cultimulchcr (2-4 times over if necessary) when soil moisture permitted. Plots were not cultivated except when weeds became a problem. In the no. tilled plots both corn and soybeans were planted with a no tillage planter.
At the North Western Branch, a Pyroxychlor (Dowco 260) treatment was superimposed on half of each tillage-rotation plot: making a total of eight treatments. Five percent granular Pyroxychlor was applied at
10 lb/A granular in the furrow at planting through an insecticide box.
A foliar spray was applied on July 3 at tho 5-6 leaf stage to the same half plots at 1/2 lb/A.in 20 gallons of water. The insecticide Furadan was banded over the corn row (at 1 lb/A a.i.) at planting time for
60 61 control of corn root worms and cut worms. Ammonium nitrate and 0-20-20 fertilizer were broadcast at 750 and 2S0 lb/A,respectively, over all plots prior to preparing seed beds. For weed control in corn after soybean plots 1 lb/A 2,4-D amino was applied 10 days prior to planting.
In continuous corn plots a mixture of Atrazino, Simazine, Paraquat,
Dicamba (1 lb/A, each) and Lasso (2 lb/A) was applied before emergence.
In the corn-soybean rotation, the above herbicides were applied at 2/3 of the above rato.
To determine the phytotoxicity levels of Truban and Pyroxychlor to com, nonstorilized kernels, 5/10 cm pot, were planted in’unsteamed Wooster silt loam and covered with vermiculite. Pots were placed in tho greenhouse (27 C) and watered daily. Pyroxychlor (1/2 lb/gal formulation) and Truban (25% B.C. formulation) were drenched, 100 ml/ pot, with five replications each at 0, 12, 25, 50, 90 and 200 ppm ug a.i./ml tho first and third weeks after planting. One month after planting, the plants wore pulled, the roots washed and root length and dry weights recorded.
To determine the effect of temperature, fungicide, and moisture on corn root rot, an experiment was conducted in growth chambers.
Soil used was a portion of the samples collected during the summer of
1975 from the tillage-rotation plots at the North Western Branch. The level of P, graminicola in soil was increased by planting crested wheat grass in previous experiments. Only soil from plots without fungicides was used. All samples were mixed, erasing the tillage and rotation variable, and placed into 35 x 20 x 15 cm black plastic containers. Two rows of five 2-kernel hills each v/ere planted in each container. Twelve containers were placed in growth chambers set at 15, 23 and 30 C and watered daily. One seedling from each hill was removed at the 3 leaf
stage leaving 10 plants/container. Moisture and fungicide variables
were imposed at the 3-leaf stage. Plants were drenched with 100 ug
a.i./ml Pyroxychlor (1/2 lb/gal formulation) at the rate of 1000 ml/
container. Wet conditions were maintained by watering daily,keeping
the soil close to saturation. Dry conditions were maintained by only
watering at tho first sign of wilting. There were three replications
of each combination of temperature, moisture and fungicide used. A
randomized-block dosign was used for the fungicido-moisture treatments.
Fertilizer (20-20-20) was applied to each containor 1 and 3 weeks after
planting. The light regime was 16 hours light and 8 hours dark. The
light intensity at tho 15, 23, 30 C was 34,000, 28,000 and 31,000 lux,
respectively. This experiment was terminated after the sixth leaf
developed.
The effects of rotation, chemical and moisture on root rot were
investigated in the greenhouse. Soil samples from the North Western
Branch from tho tilled and nontilled field plots were combined leaving
corn-corn soil and corn-soybean rotation soil separate. Soil from
tho chemical-treated plots was not used. Tho identity of tho three
field replications was maintained but a portion of each was combined
to make a fourth replication. Pooled samples were thoroughly mixed
and placed in wooden applo crates (50 x 37 x 30 cm) lined with brown plastic leaf bags. A 13 cm square opening was cut in the bottom of
the liners and covered with a 25 cm square wire screen for drainage.
Ten liters of Wooster silt loam soil were put into the bottom of tho
crates and the crates filled with the test soils (ca. 30 liters).
Treatments consisted of all combinations of two rotation variables? 63 continuous corn and corn-soybeans; three fungicide variables; no
chemical; Truban and Pyroxychlor; and two moisture variables, wet and
dry. The treatments were arranged in a complete random block design
with four replications.
Crates wcro placed under a 16 hour light (45 C), 8 hour dark
(24 C) diurnal light-temperature regime. Warm white fluouroscent
lamps producing ca. 15,000 lux were used. It was necessary to raise
the lights from time to time as the plants grew. Moisture content
was maintained in the wet treatment series by providing nine spaghetti
tubing leads/crate and watering automatically twice a day for 10 minutes.
The dry treatments were watered with ninespaghetti leads/crate whenever
the top soil appoared dry or plants showed wilting symptoms (usually
once a week).
Two kernels of hybrid corn (WF9 x OhSlA) were planted in two rows
of three hills each in oach crate. Hills were 10 cm away from the
border and 15 cm apart. Kernels were covered with 25 ml vermiculite
and watered daily until seedlingsemerged. Moisture variation was then
imposed using the automatic watoring jystem as described before. The fungicides Pyroxychlor and Truban were drenched at 100 ug a.i./ml,
3 liters/crate. For the no fungicide control, 3 liters of water/crate was applied. Treatments were applied when the seedlings were in the
3 to 4 -leaf stage. Fertilizer (20-20-20) was applied on a bi-weekly basis. Two applications ,of Monitor were applied to control black aphid infestation.
Sampling. In 1974,10 corn plants were sampled bi-weekly, early
June to mid-August from one replication each of tilled and nontilled continuous corn at the North Central Branch. In 1975, five plants 64 were sampled randomly from each of the three replications of the eight
treatments to make a total of 120 plants per sampling time from 24
plots. Sampling was bi-weekly from early June to mid-September. Both years' samples consisted of a block of soil with corn roots of about
25 cm^. In 1974,samples wore bagged separately. In 1975,plant tops were cut at the third intemode and all the five plants from each, treatment were bagged together in a 50 lb paper feed bag. Samples were moved to a cold room within 2 hours and stored overnight. The bulk of the soil was removed from the roots by hand (soil sample) and stored in the cold room until assayed for £. graminicola or used in greenhouse experiments.
In greenhouse tests, sampling was at monthly intervals. At tho first sampling two plants/crato were removed from the center 2 hills. * The second sampling was 2 months after planting when two more plants were removed from one side of tho crates, leaving two plants/crato for harvest at 3 months after planting.
Assaying Root Damage. In all those experiments root samples wore obtained by washing under a cold water jet, ca. 60 psi.
Root samples were stored at 3 C for ca. 24 hours until data were recorded. Wet weight of the root mass, including lower root bearing nodes,was obtained of the corn samples from the 1975 tillage-rotation- fungicide (TRC) experiments. All major roots were cut from the crown and five roots selected at random (25 roots/plot). The length of the selected roots and the number and type of lesion were recorded. The lesion types were: 1) small (S mm) « 2%, 2) large (6-15 mm) *= 5%,
3) streak (16-30 mm) = 10%, 4) girdle (31-50 mm) » 25%, 5 necrotic
(entire root) a ioo% of the root tissue damaged. Percentage damage 65 (root tissue with lesions) for each, treatment was calculated. Also tho percentage healthy root tissue was estimated as follows: Root length - (root length x % damage). Also, lesion types 1 and 2 wero combined as an estimate of small non-progressive lesions; and lesion types 3, 4, and 5 wero combined for largo progressive lesions.
Por sampling corn roots from the greenhouse experiment, roots were removed clockwise from the upper root whorl, then from next lower whorl until four roots/plant were obtained. Root damage was evaluated as described for tho fiold test with one exception. Throe types of girdling lesions wore recognized: 1) a girdling lesion present at tho root tips (ca. 15 cm from tho stalk) was given a rating of 1, 2) a girdling lesion 7.5-10 cm below the stalk was rated 2,and 3) a girdling losion adjacent to tho stalk was rated 3. All of tho roots except the losions plated wore dried in an oven at 100 C for 4-5 days and dry woights recorded. Other measurements and observations used to evaluate plants included: top height, root length, total number of roots, number of roots with lesions, total numbor of loaves, leaves with yellowing symptoms, dry weight of tho top and root system.
Plating, isolation and identifications of pathogens. In 1974 five root lesions from each of tho 10 plants from till and nontilled plots wero plated on Pythium selective medium (SAPBNC) as described in
Part I. In 1975 early in tho season one lesion was plated from each of the 25 roots sampled from each tillage-rotation-fungicidc plot.
Later in tho season only 10 losions wero selected from 25 roots. During the first six sampling periods roots wero plated on Pythium selective media SAPBNC only. At tho seventh sampling (September 14) one-half of each, lesion was plated on Pythium selective media (SAPBNC) and the othor half on a soil fungus medium (OAES - Part I). Six lesions were
selected from the eight roots evaluated from each rotation-fungicidc-
moisturc plot from the greenhouse experiment. From the growth chamber
experiment ono lesion was selected from each of 10 hills per container
(tcmperature-fungicide-moisturo plot). Lesions wore plated on SAPBNC,
OAES and Q 1SO4 media. Tho media used for plating and identification
are described in Part 1.
Tho corn seedling root trap (CRT) and tho creasted wheatgrass (CWG)
seedling bioassay methods wore used to estimate P. graminicola levels
in soil. In 1974, 100, 200 and 300 ml of soil removed from roots were
used for CRT assay. In 1975, two sub samples of 200 ml soil from each
treatment wero used.* Tho soil was placed in 500 ml flasks, flooded
with tap water and all floating materials removed. Five-day-old corn
seedling roots, fivo/flask, were suspended in water over soil and placed
in tho grocnhousc (1974) or growth chamber at 26 C (1975) irradiated at 28,000 lux light, 16 hours day and watered with an automatic water
sprinkler, 2 minutes duration at 2 hour intervals. The general procedure
for CWG assay was the some in 1974 and 1975 and as described in Part
II. In 1974 CWG cultivars Norlon and Intermedia were used (50 seeds/
10 cm styrofoam cups containing 200 ml soil) while in 1975 only Norlon variety was used (100 seeds/25 cm plastic pots with 1,000 ml infested soil from each of the 24 plots). Sampling, root damage estimates, and plating and isolation methods were the same as described in Part II for CRT and CWG bioassay•• Media used for plating and identification of fungi from field, greenhouse, growth chamber and bioassay methods are described in Part I. A factorial analysis of variance was computed for all variables from each, experiment. The least significant difference was used for comparison of means significantly differont at the 1% or 3% level. RESULTS
Effect of tillage, rotation, fungicides on severity of corn root rot in the field. There Wore no obvious above ground differences betweon corn in tilled (T) and nontillcd (NT) plots or continuous c o m
(CCC) and corn-soybcan-corn (CSC) rotation throughout the season at the North Western Branch. Corn in the Pyroxychlor treated (P) half of the plots was slightly stunted earlier in the season as compared to untreated plot (NP) but these differences disappeared by the first of
July. Apparently, tho level of Pyroxychlor used was slightly phytotoxic.
In general, root rot damage increased as the season progressed
(Fig. 6). There was slightly less root damage at the early August sampling. Roots sampled at this time formed during July when tho rain fall was less than normal (Table 2). Greater root damage occurred in
NT (32.0%) than in T (19.61%) and in CCC (32.09%) than in CSC plots
(19.76%). Pyroxychlor-troated plots had slightly loss damage (23.45%) than NP plots (28.36%). These differences were significant at the 1% level.
Root weights of the corn increased significantly with time P(f)»0, reaching a peak at the early August sampling (101.86 g/plant) at the same time that the least amount of root damage was evident (Fig* 6).
Subsequently root weight declined to 90.0 g/plant at the same time that root damage again increased (Pig. 7). Root weight was significantly higher in the NT plots (.79.4 g/plant) than in the T plots (72.4 g/plant).
Also the root weight was significantly less in P plots (71.4 g/plant) 68 Figure Figure Percentage root damage 6 . Mean percentage root rot dampgc of corn in field field in corn of dampgc rot root percentage Mean . plots over all tillage, rotation,tillage,all over plots fungicide and treatments during seven sampling periods in 1975.in periods sampling seven during treatments ue ue uy uy u. u. Sept. Aug. Aug. July July June June 7 0 4 8 1 25 11 28 14 30 17 Sampling time Sampling 8 70
LSD (.01)
100
* SO
30
June June July July Aug. Aug. Sept. 17 30 14 28 11 25 8 Sampling time
Figure 7. Mean root weight of corn from field plots over all tillage, rotation and fungicide treatments during seven sampling periods in 1975. 71 than in tho NP plots (80.4 g/plnnt). The root weight was not signif
icantly different in the CCC from-the CSC plots. Thus* time of
sampling had some offoct on root damage and weight. On the other hand
P-NT plots had more damage and more root rot than tho P-T plots.
Early in the season thoro wore more small lesions (types 1, and 2)
on c o m roots while at lator sampling thore were more larger lesions
(types 3, 4 and 5). Of the other factors* only P significantly affocted
the number of small lesions* (1.2/root) as compared to NP plots (1.8/
root). On the other hand P did not affect tho number of large lesions.
There wore more large losions in tho NT CO*55/root) as compared to T
plots (0.38/root), and in CCC (O.S6/root) as compared to CSC (0.38/
root) plots. Thore was a significant interaction between time of
sampling* tillage and rotation for percentage root damage. In both
rotation and tillage treatments percentage damage increased with time
except during early August when it declined (Table 9), Tho 3-way
interaction of time of sampling, tillage and rotation was not significant.
Tho tillagc-rotation* fungicidc-tillago or fungicido-rotation
interactions wore not significant for percentage root damage. ‘.This
indicates that those effects were additive.' .The greatest damage. . . occurred in the NT-CCC (38.1), NP-CCC (34.2), NP-NT (34.6) and the
least percentage damage in tho T-CSC (13.7), P-CSC (17.0), P-T (17.6) combinations,respectively. Other combinations were intermediate* varying from 22.4 to 29.8%. The tillagc-fungicidc interaction was significant* P(f)o0.15 for root weight. Tho T-P combination resulted in; significantly loss' woight (65.0 g/plant) than the NP-T (80.8 g/plant) or NP-NT (81.0 g/plant) or P-NT (80.0 g/plant). The rotation-fungicide interaction was also significant P(f)=.009, withleast root weight 72
Tabic 9. Interaction of time with tillage and time with rotation for percent root rot damage in 1975 in Hoytville soil.
Sampling Tillage Rotation time 1975 Till No-till Soybean-corn Continuous rotation corn
Juno 17 4.0a 5.5 4.0b 5.5
June 30 3.8 8.5 6.6 7.7
July 14 17.5 29.7 17.0 30.2
July 28 23.4 42.2 • 21.0 45.0
Aug. 12 17.6 37.8 21.0 35.0
Aug. 28 31.3 42.8 29.0 45.0
Sept.12 39.4 56.8 40.2 56.0
LSD (.01)=10.5
^ e a n percentage damage of five roots per plants x five plants from two rotations and two fungicides per replications with three field replications, (300 roots per sampling time).
^Mean percentage damage of five roots per plant x five plants from two tillage and two fungicides per replications with three field replications. (300 roots per sampling time). occurring in the P-CCC (67.0 g/plant) combination and most root weight occurring in tho NP-CCC (83.0 g/plant) combination. The P-CSC and
NP-CSC were intermediate. There were no significant interactions for the small lesion variable. The time-tillage, time-rotation interactions were significant at tho 1% level for the large lesions Ctypes 3, 4, S variables). The number of large lesions increased with time more rapidly in NT than T plots and in CCC than in CSC plots. The inter-, action of tillage X rotation X fungicide (TRF) was not significant for percent root damage, indicating that the effects of those three factors were additive. The percentage damage at each sampling time in the worst
TRF combination and best TRP combination is presented in Table 10. From this it was concluded that root rot was reduced substantially in plots treated with Pyroxychlor, with conventional tillage in a CSC rotation.
Tho effect of tillage, rotation, and Pyroxychlor on recovery of P. graminicola from field infected roots (Fig. 8) . Percentage root lesions
(ca. 14%) from which P. graminicola (all typos) was recovered was not significantly different in tilled vs no tilled or the CCC vs CSC rotation plots. However, only 7.8% of tho lesions in tho chemical treated plots yielded P.. graminicola as compared to 21% of tho lesions from untreated soil. Pythium graminicola types recovered were isolates forming both oospores and sporangia typical of the species, isolates forming > typical graminicola lobulate sporangia only, isolates forming miniature shrunken sporangia-like structures and isolatos form ing mycelium only on isolation plates. The latter typos formed either typical £. graminicola sporangia or oospores in grass leaf culture and all were considered to be P. graminicola. Furthermore,all were highly virulent on corn seedlings as described in Part I. As reported in 74
Table 10. Percentage root damage in tho best and worst tillage- rotation, fungicide treatments (TRF) in field plots in corn at different sampling times
Treatments Sampling time (1975)
June 17 June 30 July 14 July 28 Aug.12 Aug.28 Sept.12
TRFa 0.73° 3.20 8.70 11.90 8.40 17.00 24.80
NTRFb 11.13 9.10 40.50 56.26 51.00 54.50,. 64.86 a Conventional tillage, corn-soybean-corn rotation and Pyroxychlor applied in the furrow at planting time at 1/2 lb a.i./A b Mean percentage root damage of five roots from each of five plants from each of three replications. 75
CSC *"<-1 N« ftKfrjtwoU
Figure 8r(A to D). Field infected root systems with differences in root mass from tillage, rotation and Pyroxvchlor treated plots: A) continouous com, no Pyroxychlor; B) continuous corn, Pyroxychlor; C) corn-soybean-corn rotation, no Pyroxy- chlore; D) corn-soybean-corn rotation, Pyroxychlor. 76 Part I, recovery of P.. graminicola varied significantly with time of sampling. The time /fungicide interaction was significant for recovery of total P_. graminicola. In general, loss £. gTaminicola was recovered from lesions from chemical treatments. Of particular interest was the isolation at tho first sampling date when 3.6% of the lesions from
Pyroxychlor treated plots yielded P. graminicola, compared to 43.9% of the lesions from untreated plots. No other'Pythium spp. were recovered in sufficient numbers to detect any treatment differences.
Other fungi, predominantly H. pedicillatum and a few Fusarium spp., were also recovered from lesions using a Pythium-soloctive medium. As reported in Part I,recovery of other fungi was high when Pythium was low and low when Pythium was high (Pig. 3 Part 1).
Effect of tillage, rotation and chemical on levels of P. gramini cola in soil using seedling bioassay methods. As indicated in Part II the best methods for determining relative levols of P. graminicola in soil were to count the number of large lesions dovelopcd in the CRT assay or to count the number of crested wheatgrass soodlings surviving
2 weeks aftor planting. Also, in Part II it was demonstrated that large lesions and crested wheatgrass seedling blight were both caused primarily by P_. graminicola. The number of large .lesions Ctypes 3, 4,
5) on corn seedling roots was higher in the June sampling than in subsequent samplings (Table 11). The same trend occurred using either the number:of small lesions or percentage seedling root damage (Table .
11). Significantly fowci* large root lesions were obtained-on* corn bioassay seedlings from Pyroxychlor treated (0.42/scodling) plots than soil without Pyroxychlor (0,54/seedling). Also the number of small root lesions were fewer on bioassay tests in Pyroxychlor treated (2.9/ 77 Table 11. Numbers of small (type 1 and 2) lesions, large (type 3, 4 and 5) lesions and percentage damage of corn seed ling roots used for assaying Pythium levels An field soil at seven times during the growing season
• Variables measured
Sampling ' Percentage time Small lesionsa Large lesions** root damage
June 17 5.81C 0.60° 2S.4d
June 30 2.47 0.74 49.5
July 14 3.71 0.32 18.6
July 28 3.41 0.42 16.3
Aug. 12 3.30 0.48 18.0
Aug. 28 3.30 0.40 18.6
Sept.12 1.81 0.40 18.8
LSD (.01)= 0.8 • 0.09 6.0 a Lesions 2 mm diamoter or less. b Losions greater than 2 mm diameter. c Mean number of lesions per seedling root based on ten plants from eafeh-6f< throe replic&tionslfrom each of eight trpatmonfs. d Mean number of lesions per seedling root based on ten plants from each of three replications based on numbers and types of lesions where 5 mm lesion « 2% damage, 6-15 nun= 5% damage, 16-30 mm = 10% damage, 31-50 mm = 25% damage, entire root damage » 100% damage. 78 seedling) than in non-treated soil (3.8/scedling) and the amount of root damage was less in bioassay tests in fungicide treated' (20.6/ seedling) than non-treated soil (26.6/seedling). In summary; Pythium levels in the soil wore slightly higher early in the season and lower in Pyroxychlor treated soil. Survival of crested wheatgrass seedlings in soil sampled in June was higher (30.2 plhnts/50 seeds) than in soil samples later in the summe/ (lS;2-22,!2) -indicating'activity of P. graminicola increased in mid to late summer. This is difforent from the corn seedling root bioassay results (Table 11). However, no' differences could be detected in soil obtained from the different rotation, tillage, or fungicide treated plots. It was concluded from both bioassay tests that tho significant levels of Pythium wore present in soil throughout tho season in all treatments. Decreased root damage and poor rocovory of graminicola from field infected root losions could not be explained by the absence of £. graminicola in soil.
Effect of rotation, fungicido, and soil moisture on severity of corn root rot. Those studios were conducted in the greenhouse using field soil from the tillage-rotation plots at North Western Branch.
The soil from the tillage treatments was combined, but the identity of rotation treatment and field replication was retained. Subsequently, a moisture variable was imposed. The fungicides used were Truban (25
E.C.) and Pyroxychlor (1/2 lb/gal). In phytotoxicity tests it was found that optimum top height, root dry weight and top weight were obtained by drenching 100 ug a.i./ml of each chemical on corn. Levels of 200 ug a.i./ml of each chemical were slightly phytotoxic. One month after planting 1/3 of the plants were sampled and the roots examined. 79 Symptoms of root rot wero similar to those found in tho field plots
(Fig. 9). Effects of rotation, fungicide and moisture at three
different sampling times are summarized (Table 12, 13, 14).
The groatest rotation effect was observed at the first harvest.
Very fow root lesions were found in Pyroxychlor plots at the first
sampling but by the third sampling timcthe Pyroxychlor effect was hardly
evident. Truban resulted in significantly less root damage than no fungicide but more than Pyroxychlor at the first sampling. However, the Truban effect disappeared by the second sampling. Root damage due to Pythium was consistantly higher in the wot treatment than in the dry at all sampling times. None of the interactions were Signifi cant, except soil moisture and fungicide. Little root damage occurred in Pyroxychlor-troated wet (0.4%) or dry soils (0.25%) as compared to untreated wot (55.0%) and dry (9.7%). Significant root damage occurred in Truban-trcatcd wet (28.6%) but not dry (0.37%) soil. Tho effect of time of sampling, rotation, fungicides and soil moisture on lesion size were not as cloar cut as their effect on root damage. Tho only rotation effect evident was at tho third sampling time whore CSC had
0.49 lesions/root but CCC had only 0.15 lesions/root.
Pyroxychlor resulted in significantly fewer large and small lesions at first sampling CO.02/plant) than Truban (0.22 small losions/plant and 0.3 large losions/plant) and with no fungicide £0.25 small lesion/ plant, 1.0 large lesion/plant). There were no differences at second sampling of small root/lesions between the fungicides but Pyroxychlor again had the least number of large lesions (0.56) and no treatment had the greatest number (1.13), Differences in tho number of small or large lesions wero not evident between fungicides at sampling time 3. 80
i /
> y/'t, i r f
Figure 9-(A to F). Root samples obtained from greenhouse tost showing differences in root mass and discoloration: A) Rotation, Pyroxychlor, Wet; B) Rotation, Truban, Wet; C) Rotation, No fungicide, Wet; D) Rotation, Pyroxychlor,dry E) Rotation, Truban, Dry; F) Rotation, No fungicide, Dry. 81 Table 12, The effect of crop rotation on percent root damage in corn harvested at three different times in a greenhouse test
Rotation Sampling timoa
Qne month Two month Three month
b Continuous corn 10.9 41.4 60.5
Soybean-corn 20.5 42.3 53.7
LSD (.05) 8.27 NS NS a Each sampling time analyzed separately. b Mean of five roots from 2 plants from four replications of three fungicides and two moisture treatments. 82 Table 13. The effect of fungicides'on percent root damage in corn harvested at three different times in a greenhouse test
a Fungicides Sampling time
One month Two months Three months
Pyroxychlor 0.3b 22.4 45.1
Truban 14.5 49.6 60.2
No fungicide 32.4 53.7 66.0
LSD C.01)= 13.6 12.8 15.2 a Each sampling time analyzed separately. b Mean of five roots from two plants from four replications of two rotations and two moisture treatments. 83
Table 14. The effect of moisture on percent root damage in c o m harvested at throe diffont times in a greenhouse tost
a Sampling time Soil moisture One month Two months Three months
b d Wet 28.0 S1.0 84.5
Dry0 3.4 33.0 30.0
LSD (.01)° 11.0 9.8 18.2 a Each sampling time analyzed separately. b Watered daily to maintain soil close to saturation. c Watered whenever the top soil appeared dry or plants showed wilting symptoms ( usually once a week). d Moan of fivo roots from two plants from four replications of three fungicides and two rotaion treatments. 84 In general, there was; a larger number of both small and large lesions at all samplings in vet soil than dry soil (Table IS). One exception was at sampling time 3 where there were more small lesions in the dry than the wet treatments.
Effect of rotation, fungicide and moisture on number of types of fungi isolated from corrt roots. In the greenhouse experiment isolations were made from root lesions at all sampling dates using Pythium'select ive (SAPBNC), a goneral soil fungus (OAES) and a Rhizoctdnia selective medium (CuSO^). Rhizoctonia was not obtained at the first sampling, so, this medium was not used subsequently, lloistlxre-* had the most striking effect on tho recovery of P_. graminicola from soil.. Prom tho high moisture plots, 52% of tho lesions yielded £. graminicola as compared to 6.29% for the dry plots. Effects of the fungicide treatment were also significant with 40% recovery from root lesions from untreated soil and 10% from Pyroxychlor treated soil. Tho offocts of tho fungi- cido varied significantly with both time and moisture (Table 16).
Recovery of Pythium from roots in Pyroxychlor treated soils increased with time in the wot soil. On the other hand recovery of Pythium from untreated and Truban treated plants decreased significantly with time in wet soil. Rocovory of P;•graminicola was low at all samplings from both chemical treatments in dry soil. Pythium species,other than
£. graminicola. mostly spherical sporangial types, also wero recovered from root lesions. In general,they were affected by the time of isol- ation, fungicide, rotation and moisture factors the same as P,_ gramini cola. The Pythium-seloctive medium (SAPBNC) was also suitable for isola tion of U. nedicillatum from roots. There was no significant difference Table 15. Effect of moisture on number of small and large lesions on corn roots at three sampling times'
Sampling time
Soil One month Two months Three months moisture Small Large Small Large Small Largo lesions lesions lesions lesions lesions lesions
Weta 0.25c 0.75 0.53 1.14 0.19 1.22 n b Dry 0.28 0.16 0.25 0.56 0.45 0.82
LSD (.01)= 0.19 0.17 0.31 0.23 0.33 0.34 a Watorcd daily to maintain soil close to saturation. b Watered whenever the top soil appeared dry or plants showed wilting symptoms (usually once a week). c Mean lesion number of four roots from each of two plants from bach of three fungicides and two rotation treatments of four replications (192 roots). 86
in isolation of H. pcdicillatum with time of sampling or fungicide
treatment. More H. pedicillatum was isolated from roots in CSC than
from CCC and more from dry than wet soil. Isolation of H. pcdicillatum
was low,varying from 5.6-11.4% roots plated. Pyrenochaeta terrestris was isolated from root lesions using OAES medium. It was not recovered
at the first sampling but was? recovered from 6.9% of roots at second
sampling and 0.35% at the third sampling.
Fusarium species, primarily moniliforme, £. oxysporum, £. roseum
and £. acuminatum,also were isolated using OAES media. They increased with time significantly, varying from 27% from 1-month-old to 77% from
3-month-old plants. Significantly less Fusarium spp. were recovered from Pyroxychlor- (39%) than from Truban (55%) or no fungicide treat ments (62%). Mbro Fusarium was recovered from roots in wet (67%) than dry (39%) soil. Interaction of sampling time x fungicido x soil mois ture wore significant for recovery of Fusarium spp. (Table 16). Data for 1-month-old (first sampling) was markedly similar to the data for
P_. graminicola (Table 16) i.e. low recovery, in Pyroxychlor dronch both moistures, and high recovery in wot, Truban or no fungicide treatments.
The major difference between Fusarium and P_. graminicola recoveries from 2-and 3-month-old plants was in the effect of moisture. Pythium graminicola recovery was low and Fusarium recovery was high in low moisture soils. It was concluded that Pyroxychlor controlled Pythium in the first sampling but not subsequently. Truban controlled Pythium
* in dry soil only at tho first sampling. Pythium graminicola and Fus arium spp. were both prevalent in wet and dry soil. P. graminicola decreased with time, but Fusarium increased with time. 87 Table 16* Interaction of moisture, fungicidos and time of isola tion on recovery of Pythium graminicola and Fusarium spp. from diseased roots iji the greenhouse
P. graminicola Fusarium spp • •
Sampling time (month) Sampling time (month)
Treatments 1 2 3 1 2 3 • Pyroxychlor Wet 2.0b 33.3 56.2 0.0 33.3 97.9
Dry 4.2 8.3 2.0 0.0 47.7 56.2
Truban Wet 7S.0 56.2 35.4 56.2 72.9 93.7
Dry 0.0 4.1 10.2 2.0 33.3 70.8
Nothing Wet 100.0 66.7 45.8 75.0 75.0 75.0
Dry 18.8 0.0 8.3 27.0 47.9 70.8
LSD (.01)= :28.18
One, two or three months after planting. b Moan percentage of lesions based on three roots from two plants from four replications from two rotations (48 roots/sampling timo/ treatment). 88 Effects of temperature, moisturo and Pyroxychlor on corn root rot.
These experiments were conducted using £. graminicola-enriched soil
in small containers in growth chambers. Plants were harvested, roots washed and data taken at the 6tli. leaf stage. Large differences in root jpass were evident among moisture and fungicide treatments at 23 C.
(Fig. 10). The greater number of roots were found at 30 C (3.S/plant)
and least at 15 C (3:1/plant). Roots in the .high moisture treatment had 6.1 infected roots/plant compared to 2.1 in the dry treatment. In
Pyroxychlor-treated plots there were 3.5 infected roots/plant as comp ared to 4.8 in the untreated plot.
Tho interaction of temperature and moisture was significant
(Table 17). In wet soil there was a significantly greater number of infected roots at 30 C than at either 23 C or 15 C. However, in dry soil thore wero significantly fewer infected roots at 15 C than at either 23 C or 30 C. The temperature moisture and fungicide interact ion was not significant indicating that tho fungicide effect was addit- . ivo to the temperature moisture interaction.
Pythium graminicola was tho only Pythium spp. isolated from the infected roots. It was not recovered from roots in the Pyroxychlor treated plots. This pathogen was recovered most frequently (56.6->
* 93.3%) ■ from high moisture untreated plots .at all 3 temperatures. Some*
£. graminicola was recovered from the untreated dry soil at all 3 temperatures (13.3-40.0%). In contrast,H. pedicillatum was recovered
* most frequently from wet (84%) than from the dry (66%) soil. It is concluded that high temperature and high, moisture are conducive to corn seedling root rot caused by these pathogens. Also, root rot was reduced significantly by a soil treatment with Pyroxychlor, Figure 10-(A to D). Root samples from growth chamber experiment showing differences in root mass and discoloration: A) 23 C, Dry, Pyroxychlor; B) 23 C, Dry, No Pyroxychlor; C) 23 C, Wot, Pyroxychlor; D) 23 C, Dry, No Pyroxychlor. 90
Table 17. Interaction of temperature and moisture on number of infected corn roots in growth chamber tests
Temperature • (C) Moisture
b Wota Dry
15 S.3C 0.9
23 4.7 2.9
30 8.5 2.5
LSD C‘01)= 0.64 a Watored daily to keep tho soil close to saturation. b Dry conditions were maintained by only watering at the first sign of wilting. c Mean number of infected roots for ten roots per plant from ten plants per container from six containers per temperature.
4 DISCUSSION
In this study tho effects of time of sampling, tillage, rotation, fungicides, moisture and temperature on corn root rot and occurrence of
Pythium graminicola wore investigated. In general, percentage root damage increased with timo in the fiold without any obvious recogniz able aerial symptoms. There was slightly less root damago at the early
August sampling. Decline in root damage, probably, was the result of rapid development of new roots when soil moisturo lovels were too low for £. graminicola to cause infection evon though it was present.
Greater damago occurred in plots without tillage and rotation.
Tillage and rotation differences in root damago cannot be explained by differences in levels of £. graminicola. Nontilled cropping in which root rot was worst probably creatod conditions conducive to root rot.
Therefore, greater root damago occurrs even though Pythium levels may not be excessively high. It is not yet known what these conditions are. It is ovident that a no-tillage system conserves water and may increase root development near the soil surface. Triplett et al. (91) reported that a no-tillage system with mulcfi cover reduced evaporation and conserved water. Also, the influence of cropping scquenco on crop yields is affected by tillage and drainage. There is a tremendous potential for no-tillage corn in Ohio. Triplett et ai. (91) have suggested that no-tillage corn production systems should be located primarily on the well drained, sloping soils in Ohio. No-tillage 91 92
treatments have suffered substantial yield losses on some poorly-
drained soils (22, 23).
The increased damaged from cropping corn continously cannot bo
explained by an increase in amount of P_. graminicola. Pythium gramini
cola level was slightly higher in CCC than CSC, but not significantly
;so. Perhaps other factors are involved, i.e. greater activity of
i inoculum on corn as opposed to soybean residue which could bot be dete
cted using the bioassay method. Many investigators (41, 66,.109, .110)
have reported that crop rotation with soybean is bonbficial to corn.
Although yields were taken in this investigation there were no
significant differences because the stands were depleted by repeated
sampling that resulted in extremely high variation between replications.
However, Van Dorcn et al. (99) have roported a significant tillage-and
♦ rotation interaction in the same plots over a ten year poriod. Lowest
yields occurred in Ho-till (NT)- Continuous corn (CCC) while tho othor
combinations yielded 720 to 1180 kg/H more corn grain. Williams and
Schmitthonnor (109) also reported significantly higher yiolds in
Corn-soybean-corn (CSC) than CCC rotations. It is highly probable
that decrease in yield is the result of increased root damage initiated
by £. graminicola and perhaps inhanced by other soil microorganisms.
Additional evidence that £. graminicola was tho primary cause of
corn root rot was obtained in tests with Pyroxychlor and Truban, both
Pythium selective fungicides. In a field experiment, Pyroxychlor
* significantly reduced root damage.. There were significantly fewer small
lesions (types 1 and 2) early in the season in Pyroxychlor treated
plots. Also, Pyroxychlor treated plots had a lower level of p. gram-
inicola than in the untreated soil. This would indicate that fungicide Pyroxychlor probably protected corn roots from Pythium and the latter vraj responsible for the small lesions. Later in the season there were more large than small losions; Pyroxychlor had no effect on tho number of large lesions. Apparently, it lost its effect in about 6 weeks.
However, Pyroxychlor-treated plant had a reduced root weight as comp ared to untreated plants. Also, early in the season Pyroxychlor- treated plants were slightly stunted. Evidently,this compound is phytotoxic at concentrations of 1/2 lb a.i./A when applied in the ' furrow.. Control of Pythium may have been offset by its phytotoxicity.
Alternative methods need to be developed for applying Pyroxychlor to corn to avoid phytotoxicity but still control Pythium. It might then be possible to use Pyroxychlor to determine tho effect of Pythium root rot on corn yield.
Tho effect of cither Pyroxychlor and Truban on corn root rot *was ; studied in tho greonhouso along with moisture and rotation factors,
Using soil from tillage and rotation field plots. Both Pyroxychlor and TrUban reduced root damago and number of lesions on plant roots samplcdcne month after planting. Pyroxychlor was effective in both wet and dry soil, whereas Truban was effective only in dry soil, particularly during first sampling. Two months after planting neither fungicide was effective (Table 16). Recovery of P_. graminicola from lesions on Pyroxychlor treated roots in both wet and dry soil was low one month after planting. Rocovery was high from roots from Truban treated wet soil and low in Truban treated dry soil. Effect of moists uro on both root damago and recovery of JP. graminicola was highly significant. This moisture relationship and Pythium activity is in agreement with the findings of McLaughlin (52). In general, recovery 94 of Fusarium and other fungi was high, when Pythium was low and low when pythium was high. This is evidence that there is a strong association between Fusarium and Pythium. Probably Fusarlum sp. aro probably secondary invaders in Pythium induced lesions, especially early in the season. However, Fusarium sp-. may be primary invaders of scenesccnt roots.
Additional data on the effects of Pyroxychlor and moisture were obtained in growth chamber experiments where an additional factor, temperature was also included. There were no significant interaction between fungicide, moisture and temperature indicating that these factors were also operating independently and were additive. In this experiment there were significantly fewer root lesions in Pyroxychlor treated soil. There was no reduction in root weight in Pyroxychlor treatment either in tho greenhouse or growth chamber experiments, indicating that this compound was not phytotoxic when applied as a
100 ug a.i./ml drench early in the season. This method of application possibly could be used for evaluating effects of Pythium on corn yields. The effects of moisture on root rot also wore very striking in growth chamber as well as greenhouse experiments. Good control of
Pythium root rot can be obtained in dry soil. Unfortunately this approach, to control is not feasible in high, rainfall areas, but might be considered in irrigated c o m in the West. This relationship of high, soil moisture to Pythium root rot damage might explain the increase in root damage in nontilled as opposed to tilled soil. Nontilled soil might retain moisture because of less evaporation from mulch cover.
Also, in poorly drained, nontilled soil, noncapillary water might be retained longer because the upper 20 cm is more compacted than in 95
tilled soil.
Surprisingly, tho greatest amount of root damage occurred at
30 C in the growth chamber. Pythium graminicola recovery from root
lesions also was higher'. Increased root damage in nontilled soil
could not bo explained on the basis of it being cooler than tilled
soil because of a mulch cover. However, the interaction of moisture
and temperature was significant. Apparently, high tcmpcraturo only
effects root damage at high moisture levols (Table. 16). Based on this
information it is possihle that severe Pythium root damage could occur
in corn irrigated in mid-summer or after excessively rainy periods in midrtsummor. From these experiments it can be predicted that the worst
Pythium damage would occur in continuously cropped nontillcd corn or during a rainy summor or when temperatures reach high levels. Fungi cides liko Pyroxycholor might control Pythium even under those optimum root rot conditions if they can bo applied several times during the season and in a manner that would not be phytotoxic. A number of aspects of corn root rot -fleed to bo.investigated: factors like ago of plant, plant population density, effect of tempera ture and moisture, and interation of microorganisms and virusos which may influence the susceptibility of c o m plant to root rot. Based on the preliminary work conducted in the greenhouse, it appears that corn becomes less susceptible to Pythium with age. The physiological con dition of the plant might have a role in susceptibility. However, in the field changes in maturity of the corn are confounded with, moisture variables. Hampton and Buchholtz (25) reported that moisture and temp erature were responsible for the absence of P. graminicola from corn roots. In this study it was found that £. graminicola recovery declined as tho plant matured, even though the plots were maintained close to saturation. From this it was concluded that young plant roots are susceptible to £. graminicola but during the rapid vegetative development phase of the plant the root system probably is more resistant. However, lesions and P. graminicola reappear late in the season when plants are in the reproductive phase. Thus there seems to be host physiological factors that are responsible for data obtained for P.. graminicola recovery and susceptibility of corn roots.
It was possible to differentiate the difference•in age (phy siological state) of the plant and the offect of moisture in the greenhouse tests. But more work is needed to determine factors involved in inducing susceptibility of corn to root rot under field conditions. In the field tests it was not clear what role a decrease in moisture as the season progressed might have in decreasing Pythium in roots. The moisture effect could be studied in tho field if irrigation was available. More information is also needed regarding the occurrence and prevalence of all fungi in roots thoughout the season. Pythium was high when other fungi were low and other fungi were high when Pythium was low. It is not known if other fungi, i.e.
Fusarium, Helminthosporium and Pyrenochaeta, inhibit Pythium in losions or interfere with tho recovery of Pythium from lesions. Recovery of
"w P. graminicola from soil using CRT or CWG bioassay methods can be used to screen large numbers of soil samples for t\ graminicola and their virulence to corn. Using CRT and CWG methods or Petri dish water culture seedling pathogenicity tests a large number of different corn lines could readily be screened for disease resistance. 97 More work, is needed to understand the relationship of root rot to stalk rot. The corn culturing procedure adopted in the greenhouse
study night be useful in elucidating this complex disease problem.
There is very little information available regarding the relation ship of root rot to yield loss; Therefore, it might be possible to use the c o m culturing method adopted in the greenhouse to control
Pythium and to obtain preliminary information regarding the relation ship of root rot to yield.
The concept and methods developed in this investigation to control the primary pathogen, P.. graminicola, might be adapted to field tests to determine the relationship of root damage to yield. SUMMARY
•
This study was undertaken to determine the causual agents of root
rot associated with yielJ reduction in nontilled corn in heavy clay
soil in Ohio and to determine tho effects of tillage, rotation, fungi
cides, temperature, and moisture on levels of thcso causal agents and
severity of root rot. Corn root rot has been given little recognition
in recent years, even though, it has been reported and studied by many people in the C o m Belt and in many of the corn-producing countries of
tho world. Root rot of corn in North Western Ohio first appeared as
small, yellowish-brown lesions on primary and seminal roots early in
the season. Those lesions enlarged and coalesced, resulting in a necrotic and black root system by the end of the season. In some
instances, the root system was so severely rotted that the c o m plants
lodged.
During the summer of 1974 and 1975, biweekly isolations from corn roots from two long-term tillage-rotation experiments in heavy clay soil wore made on a Pythium-solective medium. Pythium graminicola was found early in tho season in both years and also late in tho season in
1975. This pathogen caused extensive root necrosis and reduced root mass in corn seedling pathogenicity tests. Pythium torulosum and IP. dissotocum were isolated frequently early in the season and were only slightly virulent. Helminthosporium pedicillatum was isolated through out the season on the Pythium-selective medium. It produced occasional
98 dark lesions but did not reduce the root mass in the pathogenicity
tests. Late in the season Fusarium spp. wero most prevalent; H.
pediclllatum and Pyrenochaota terrcstrls wore less prevalent, and
Trichodcrma, PeniclIlium, and Mucor&les wore isolated infrequently
on a nonsclectivo medium. In seedling pathogenicity tests, £. oxysporum
and F_. moniliformo reduced'root development without obvious root rot,
suggesting that they formed materials toxic to corn roots. Fusarium
roscum and JP. acuminatum were not pathogenic and did not reduce root
mass. Pyrenochaota terrostris caused a red root rot with scattered
lesions, but did not reduce root growth. In combination studios
wherever P. graminicola was present, root rot damage was very severe.
None of tho other fungi interacted synergistically with Pythium or
protected roots from Pythium damage.
In a greenhouse study using field soil, tho samo fungi wero
isolated as in the field study with P,. graminicola recovered most
frequently from 1-month old plants, species of Fusarium and Pyrenochaota
most frequently from 3-raonth old plants, and H. pedicillatum at low
frequency at all ages. It was concluded that JP. graminicola was tho
primary incitant of corn root rot in young plants (1 month) in the
soil tested. Other fungi isolated probably were weak parasitos and
secondary invaders of primary incitants of root rot of senescent plants.
A corn seedling root trap (CRT) method was developed for detection
of zoospore-forming species of Pythium in soil that were pathogenic to
corn roots. This method was compared with, a crested wheatgrass bioassay
(CWG) using Norlon and Intermedia cultlvars. Only P. graminicola was isolated from large, progressive lesions. On corn seedling roots or from diseased crested wheatgrass seedlings. The Norlon cultivar was 100 the most susceptible. Relative levels of JP. graminicola could be evaluated by the amount of damage or number of large lesions on seedling roots in the’CRT assay or by'the number of crested wheat- gross seedlings killed in the CWG method.
The effects of tillage (tilled vs. nontilled), rotation (continuous corn vs. soybean-corn),' and a soil fungicide (Pyroxychlor at 1/2 lb a.i./A) on the severity of root rot and levels of £. graminicola in soil was studied in a long-term tillage-rotation experiment sampled in 1975.
In general, root damage increased as the season progressed. Least root damage occurred in the tilled soil, corn-soybean rotation, and
Pyroxychlor treated plots. Interaction of tillage, rotation and fungicides was not significant. Therefore, these factors were additive.
The best root development occurred in rotated corn-tilled-Pyroxychlor treatment, and most root rot occurred in the continuous corn-nontilled- no Pyroxychlor treatment.
Pythium graminicola and other Pythium spp. wero recovered from corn roots in the field in equal amounts from tilled and nontilled and continuous corn and com-soybean treatments; but less frequently from roots from Pyroxychlor than from no Pyroxychlor treatments. Pythium graminicola was isolated most frequently from corn roots in the field early in the season, but least frequently during late July and August when root damage was increasing.
Results from the CRT and CWG hioassay indicated that significant levels of P. graminicola were present in soil throughout the season, although the levels decreased slightly as the season progressed.
Pythium graminicola levols were not affected by rotation and tillage 101 treatments, but were reduced slightly by Pyroxychlor. It was. concluded that differences in recovery of P.. graminicola from damaged roots in the field could not bo explained'by differences in levels of £. graminicola in soil.
Tho effects of cropping history, fungicides (100 VS a.i./ml drench) and soil moisture were evaluated in a greenhouse experiment using fiold soil. C o m was sampled at 1, 2, and 3 months after plant ing. Moro root damage occurred at all sampling times in saturated soil than in soil watered just enough to prevent wilting. Root damage was less severe in both Truban- and Pyroxychlor-drcnchcd soil than in untreated soil 1 month after planting. Two months after planting only Pyroxychlor-treated soil showed less damago than tho untreated soil. Three months after planting, root damage was also evident in the Pyroxychlor-drcnchcd soil. Again, thore was loss root damage in soil from corn-soybcan rotation field plots than in soil from continuous corn field plots. Pythium graminicola was isolated from damaged roots most frequently from the saturated soil-no fungicide or saturated soil-Truban treatments. It is concluded from this experiment that either low moisture or Pyroxychlor significantly reduced root rot damage and provented P.. graminicola infection of c o m roots. Fusarium spp. were the predominant fungi isolated from lesions found in Pyroxychlor-drenched.soil. Pythium graminicola was isolated from lesions in Truban-drenched soil.
Recovery of Fusarium was similar to P_. graminicola at the first samping time; low in Pyroxychlor, higher in Truban and highest in non fungicide treatments. Subsequently, Fusarium recovery was high, in both wet and dry treatments, whereas recovery of £. graminicola was low in 102 dry treatments.
The effects of temperature, moisturo and fungicides on severity of seedling root rot was studied in growth chambers using field soil.
Root rot was most severe at 30 C, least at IS C, and intermediate at
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