I This dissertation has been microfilmed exactly as received 66-10,031

MWANZA, Nelson Peter, 1937- VIRUSES AS PREDISPOSING FACTORS IN THE SUSCEPT­ IBILITY OF CORN AND PLANTS TO OTHER PATHOGENS. The Ohio State University, Ph.D., 1966 Botany

University Microfilms, Inc., Ann Arbor, Michigan VIRUSES AS PREDISPOSING FACTORS IN THE SUSCEPTIBILITY OF CORN AND WHEAT PLANTS TO OTHER PATHOGENS

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

Nelson Peter Mwanza, B.Sc.(London)} M.Sc.

*******

The Ohio State University 1966

Approved by

/ / Ci-U

I am indebted to Dr. L. E. Williams for aid and en­ couragement throughout the course of the research and prep aration of the dissertation and to Dr. C. W. Ellett for reviewing the dissertation and offering helpful criticisms Thanks are expressed to the faculty and fellow graduate students of the Department of Botany and Plant Pathology for their helpful suggestions and assistance. Grateful thanks also are due to the Ohio Agricultural Research and

Development Center for providing some of the materials used in the research. CONTENTS

Page

INTRODUCTION...... 1 LITERATURE REVIEW...... 3 MATERIALS AND METHODS...... 6 The viruses ...... 6

The f u n g i ...... 7 Inoculation of corn and wheat plants with viruses 7 Inoculation of corn and wheat plants with fungal and bacterial pathogens ...... 8

Disease index or rating method...... 9 EXPERIMENTAL RESULTS ...... 12 The effect of MDMV on the susceptibility of corn plants to different root rot pathogens...... 12

The effect of time of inoculation of corn with P. graminicola and MDMV on root rot severity. . . lo The effect of temperature on development of root rot in virus-infected and virus- free corn plants...... ' ...... 18

The effect of inoculum level of P. graminicola in inducing root rot in virus-infected and virus- free corn plants...... 20 The rate of root rot development in virus- infected and virus-free plants at normal level inoculum of P. graminicola...... 23

The effect of 3A virus on the susceptibility of corn and wheat to root rot caused by P. grami­ nicola ...... 2 6

iii C ONTENTS-- (Continued)

Page

The effect of MDMV on the susceptibility of corn plants to seedling blights caused by various organisms...... ■...... 27 The effect of MDMV on the susceptibility of corn plants to stalk rot...... 30

DISCUSSION...... 33 SUMMARY...... 36 LITERATURE CITED ...... 38

iv. TABLES

Table - Page 1 Mean root rot indices in MDMV-infected and virus-free corn plants inoculated with various fungi, ...... 13

2 Root rot indices in virus-infected and virus- free corn plants inoculated with P. graminicola at varying intervals...... 18 3 Effect of temperature on root rot development caused by P. graminicola in virus-infected and virus-free corn plants...... 19 4 Percentage of wilted corn plants when inoculum level of P. graminicola was doubled (1/2 plate/ pot)...... 23 5 Effect of 3A virus on the susceptibility of corn and wheat to Pythium root r o t ...... 26 6 Stalk rot indices in virus-infected and virus- free corn plants 12 days after inoculating with f u n g i ...... 31

v FIGURES

Figure Page 1 Root rot index scale ...... 10 2 Stunting and wilting symptoms caused by _P. grami­ nicola in virus-infected and virus-free plants . 15 . 3 Root rot development 10 days after inoculating virus-infected and virus-free corn plants with P. graminicola...... 17 4 The effect of increased inoculum (1/2 plate/ pot) of P. graminicola on virus-infected and virus-free corn plants ...... •. 21 5~~The rate of development of ]?. graminicola root rot in virus-infected and virus-free corn p l a n t s ...... 25 6 Percentage of seedling blight due to different pathogens in virus-infected and virus-free corn plants...... 29

7 Seedling blight caused by Penicillium purpuro- genurn in virus-infected and virus-free corn seedlings...... 32

vi INTRODUCTION

Corn stalk and root rots are complex problems. The causal agents of such disorders may be difficult to isolate for study. Frequently a multiplicity of pathogens, some of which appear to be unrelated to the problem, are in­ volved. Some of the fungi frequently isolated are species of Fusarium, Cephalosporium, Penicillium, Stemphylium, and Helminthosporium. Although some of these are not usually pathogenic to corn under normal conditions they should not be ignored since they may become destructive when the sus­ ceptibility of corn is increased under unusual conditions.

There has been mounting evidence that the susceptibility of plants to disease development may be influenced by the nutrition of the host. Recently,, there is evidence that viruses also affect the susceptibility of plants to other pathogens.

It was observed in the field that corn plants infected with dwarf mosaic virus were more subject to root and stalk rots than virus-free ones (22). Similar observations were made in which corn infected with corn stunt virus de­ veloped basal rot caused by Fusarium spp. which were not pathogenic to virus-free plants (6 ).

1 Viruses increasing the susceptibility of plant to

/ other pathogens could create serious problems in the pro­ duction of crops in an area where the use of disease tol­ erance in a breeding program is practiced. Yield of the host crop would be reduced not only due to virus infection per se but also to increased susceptibility of the plants to pathogens and secondary pathogens that might not have any significant effect without the presence of the virus in the host. In this research, the effect of maize dwarf mosaic virus and 3A virus on the susceptibility of corn and wheat to other pathogens was investigated. In the past, several of the investigations on the effect of viruses on the susceptibility of plants were of a preliminary nature.

The intent of this work was to conduct investigations which would yield more quantitative interpretations. Sev­ eral organisms, ranging from pathogenic to non-pathogenic under normal conditions were used. LITERATURE REVIEW

The susceptibility of corn and wheat plants to root rot, stalk rot, and seedling blight pathogens is affected by many factors. Some of the factors are the age of the ■ host plant, the environmental temperature, soil moisture, and nutrition of the host. Many workers have investigated the effect of mineral nutrition on the susceptibility of corn and other plants to root rots, stalk rots, and other diseases (1,2,5,10,13,16,17,19*20). Most of the workers agree that deficiency, excess or imbalance of certain elements increases the susceptibility of the host plant to pathogens, but the proper amounts and ratios of the mineral elements decreases the. susceptibility. It seems that the increased susceptibility is the result of unbal­ anced metabolism resulting from the deficiency or imbal­ ance of the elements.

Viruses also have been reported to affect the suscep­ tibility of plants to other pathogens. A few investigations have been made on this phenomenon. Thomas and Bov/man (18) found that oat plants infected with red-leaf virus developed root rots caused by root rot fungi'Which did not affect . _ virus-free plants. . The root rot was more severe under con­ ditions of low soil temperatures. Smith (15) reported that 4 yellow dwarf virus (BYDV) played a role as a pre­ disposing factor in the common root rot of wheat in Canada and New Zealand. His observations, both in Canada and New

Zealand, indicated that Fusarium and Rhizoctonia species became pathogenic predominantly on wheat plants infected with BYDV. However, the phenomenon did not apply to dis­ eases caused by Ophiobolus graminis and Cercosporella her- potrichoides, both of which caused root rot disease of wheat independently of BYDV infection in New Zealand. Forrest, Hensely, "Candia, and Pi rone (6 ) found that the incidence of basal stalk rot in corn plants stunted by the corn stunt virus was greater than in plants not showing virus symptoms. Isolations from basaliy rotted plants yielded a Fusarium sp. which failed to induce rot in healthy plants. The investigators thought it probable that invasion by this and other pathogens was favored by the weakening of the plant by the stunt inducing agent. Further work in the virus-fungus relationship affect­ ing the development of root rots of was done by Farley and Lockwood (3*^)• In greenhouse tests with the mosaic virus (PMV) and • Aphanomyces ..euteiches and Fusarium solani f. Pisi, they found that root rot disease indices in the plants with combined virus-fungus infection were twice that..in the plants inoculated with fungus only. Watson

(21)also reported a virus-fungus relationship in a root rot complex in red clover. Plants infected with clover yellow mosaic virus showed more severe root rot than the

virus-free plants. Fungi which were most frequently iso­ lated were Fusarium oxysporum3 F_. roseum, and Tetrac oco- sporium paxianum which were only mildly pathogenic to red clover plants without the'clover yellow mosaic virus.

Williams and Alexander (22) observed that root rots and stalk rots were more severe in corn plants that showed maize dwarf mosaic symptoms. Pythium graminicola Subrm.3 Rhlzoctonia solanl Kuehn3 Helminthosporlum pedicellatum Henry3 and several Fusarium spp. were isolated from the roots. MATERIALS AND METHODS

Three varieties of corn (Zea mays L.), yellow dent corn, Ohio ¥-49, Moews MCB98¥ and yellow sweet corn, Golden Bantam, and Lucas wheat (Triticum aestivum L.), were used. Plants were grown in steamed mixture of three parts wooster silt loam and one part muck. The soil was then placed in sterile plastic_ pots (4 inch X 4 inch) perfor­ ated at the bottom for drainage. Four to six seeds of host plants were planted in each pot. Most of the inves­ tigations were carried out under a greenhouse temperature of 75°F to 90°F. In summer, an evaporative cooler was used. Some work was carried on in constant environment rooms.

The viruses

Two viruses were used: maize dwarf mosaic virus (MDMV), which is mechanically transmissible to corn and several other monocotyledonous hosts but not to wheat, and a virus • (called 3A) mechanically transmissible to both corn and wheat. 3A virus also differs from MDMV on the basis of serology and incubation period ( 2 3 ) There is strong belief that 3A virus may be the wheat streak mosaic virus (WSMV). 7

The fungi

/• All the fungi used were from stock cultures of L. E. Williams, Ohio Agricultural Research and Development Center. All xvere .previously isolated from corn plants. They were maintained on potato dextrose agar (PDA). For pathogenicity tests 20 ml. of the medium was used in sterile petri plates. After-the medium had cooled and solidified, a 2 mm. disc of the mycelium of the test organism was placed in each petri plate and incubated at 25°-28°C for specific periods. The following fungi were used: Cephalosporium sp., Dlplodla zeae (Schw.) Lev., Fusarium moniliforme (Sheld.) Snyder and Hansen, Fusarium oxysporum Schlecht., Fusarium roseum f. cerealls (Cke.) Snyder and Hansen, Helminthosporium pedi- cellatum Henry, Penicillium funiculosum Thom., Peniclllium oxalicum Currie and Thom., Penicillium purpurogenum Stoll, Pyrenochaeta terrestris (Hans.) Gorenz, J. C. Walker and Larson, Pythium graminicola Subrm., Stemphylium sp. Bacter­ ium stewartii E. F. Sm., which the author isolated from corn plants with Stewart's disease symptoms, was included. It was cultured on yeast extract glucose agar. Twenty ml. of the medium were used in each-petri plate. Plates were streaked with the bacterium and incubated at 25°-28°C.

Inoculation of corn and wheat plants with viruses The viruses used were maintained in corn and wheat growing in the greenhouse. Both hosts were inoculated with virus at the second leaf stage, approximately 8 days after planting. Pots with seedlings of uniform height and size were selected to reduce variability. Virus inoculum was

obtained by grinding virus-infected leaves with a 0.005 M neutral phosphate buffer in a mortar. Buffer and leaves were mixed 5 :1 * For inoculations virus extract was rubbed gently on the second leaf 'of each plant previously dusted — with 600 mesh carborundum. Inoculated plants were then placed in a moist chamber, for 2k hours, in which relative humidity was maintained near 100 percent. Control plants were similarly dusted with carborundum but rubbed with virus-free buffer extract from plants. Virus symptoms

in inoculated seedlings became apparent on the third day after inoculation.

Inoculation of corn and wheat seedlings with fungal and bacterial pathogens

For fungal root rot and seedling blight studies, two

inoculation methods were used. The first was described by Schmitthenner and Hilty (14) in which the bottom 1/3

of the soil in the pot was mixed with the sliced up fungus and then the host plant seeds were planted on the top layer of the soil. As the roots developed they encountered the pathogen already present and spreading in soil. The second was a modified Hoppe's "mycel mat method" (7). With this, the mycelial mat was removed intact from a plate and placed 9 either at the bottom or center of the pot in which the plants were already growing. This method had the advan­ tage of allowing the plants to be rooted, thus making it possible to inoculate them with virus previous to the fungus.

The inoculum level in case of £. graminicola, which was very pathogenic to corn and wheat, was usually 1/4 of a 6 -day-old PDA. plate culture per pot. The other fungi were used at four times this amount.

Corn stalk inoculations were made 7-8 weeks after planting by a cork borer method described by Williams and Menon (24). Plants were inoculated with 13. stewartii by injecting a 1 ml. suspension of cells in sterile distilled water into 8 -day-old plants with a hypodermic syringe. In some tests plants were inoculated by the clip method (8 ) in which the leaves were clipped with a sterile pair of scissors and the clipped ends swabbed with cotton dipped in the bacterial suspension.

Disease index or rating method

Root rot severity was determined on the basis of per­ centage of the adventitious roots with dark or brown lesions. Test plants were removed from the pots and immersed in a bucket of water and left for at least 6 hours when most of the soil had loosened and roots needed only slight rinsing for observation. Extent of root rot was rated on the scale:

0 = no rot; 1 = trace to 25 percent of roots with lesions; Pig. 1. Hoot rot index scale showing: 0=no root rot; 1= trace to 25 percent of roots with lesions; 4=76 to 100 percent of roots with lesions. 11

2 = 26 to 50 percent of roots with lesions; 3 = 51 to 75 percent of roots with lesions; and 4 = j 6 to 100 percent of roots with lesions. Dead plants were separately men­ tioned in the results but calculated as class 4. The severity of stalk rot was rated on the scale: 1/2 slight necrosis around the point of inoculation; 1 = trace to 25 percent of inoculated internode rotted; 2 = 26 to 50 percent of inoculated internode rotted; 3 = 51 to 75 percent of inoculated internode rotted; 4 = 76 to 100 percent of inoculated internode rotted; and 5 = rot extending beyond the inoculated internode. Dead plants were included in class 5.

Seedling blight and wilt were determined on the basis of percentage of plants blighted or wilted. EXPERIMENTAL RESULTS

The effect of MDMV on the suscepti- billty of corn plants -to differerrT root; rot' pathogens

Since several workers have reported that fungi which caused disease on virus-infected plants did not cause dis­ ease in virus-free plants (6 ,15,18), the first experiments in this work were designed to find out if MDMV-infected corn plants would be more susceptible to pathogenic and normally non-pathogenic fungi.

In separate experiments, MDMV-infected and virus-free corn plants were inoculated with 12 different fungi (Table 1). Ten days after inoculation, data indicated that Pythium graminicola, Dlplodla zeae and Fusarium moniliforme caused more root rot than the other fungi. The root rot was sig­ nificantly greater in virus-infected plants than in virus- free plants. It was interesting-to note from the data that D. zeae and F. moniliforme, which caused almost negligible amount of root rot in virus-free plants, caused as much root rot in virus-infected plants as that caused by P. grami­ nicola, which is a virulent pathogen of corn, in virus-free plants. Fusarium oxysporum, F. roseum, Helminthosporium pedicellatum and Stemphylium sp. caused a relatively small amount of root rot in either virus-infected or virus-free

12 13

Table 1. Mean root rot indices in MDMV-infected and virus- . free corn plants inoculated with various fungi.

Root rot index3.

Pathogen Virus-free Virus-infected

Cephalosporium sp. 0.02 0.02 Diplodia zeae 0.26 2.01**

Fusarium moniliforme 0.50 1.47** F. oxysporum 0.06 0 .70**

F. roseum 0.17 0.37* Helminthosporium pedicellatum 0.24 0.79** Penicillium funiculosum 0.02 0.02 P. oxalicum 0.03 0.04 P . purpurogenum o.o4 0.05 Pyrenochaeta terrestris 0.02 0.02 Pythium graminicola 1.46 2 .92** Stemphylium sp. 0.52 0.63

aSach index was a mean of 6 plants in each of the 6-7 replications. •^Significant at 0.05; **sighificant at 0.01 level. All control plants had a mean root rot index of 0.0. plants, but the rot was greater in virus-infected plants except with Stemphyltum sp. Cephalosporium sp. and

Pyrenochaeta terrestris caused only slight lesions on some of the plants. Seedling blight was observed in experiments with Fusarium moniliforme, F. roseum, and Penicillium spp. especially in the virus-infected plants. Repeated experi­ ments gave similar results as those recorded in Table 1.

Since it was observed that virus-infected plants de­ veloped more blight than the virus-free plants (as in root rot) it was desirable that investigations on disorders other than root rot be included. Also, because P.- grami­ nicola caused the most severe root rot, stunting and wilting (Fig. 2) which were more apparent in virus-infected plants,

it was selected for further studies on the effect of viruses on the susceptibility of corn and wheat plants to root rot or wilt development. 15

Fig. 2. Stunting and wilting symptoms caused by P. grami­ nicola in virus-infected and virus-free young corn plants. £ = untreated plants; B = inoculated with MDMV; C = inoc­ ulated with P. graminicola; D = inoculated with MDMV and P. graminicoTa. 16

The effect of time of inoculation of corn with P. graminicola ancT MDMV on root rot severity

In investigating the effect of viruses on the suscep­ tibility of corn and wheat to other pathogens, plants were inoculated with virus as early as possible then fungal in­ oculations followed 2 days later. This practice was adopted after a series of tests in which the virus inoculation of corn was done 8 days after planting (second leaf stage) and inoculation with P. graminicola was at the time of planting, concurrently with, then 1 , 2 , 3 , and 4 days after virus inoculation as indicated in Table 2. The data (Table 2) indicated that virus infected plants rated significantly higher in root rot index than the virus-free plants except where fungal inoculation was at seeding. Plants inoculated with P_. graminicola at seeding developed early root rot and inoculation with virus later made little difference to the subsequent root rot develop­ ment. Allowing the plants to become rooted and inoculating them with virus before P. graminicola was therefore a better way of studying the effect of MDMV on the susceptibility of young corn plants to root rot. In a repeated experiment similar results were obtained except when inoculation was done on 0 day when virus-free plants gave a higher root rot index than the one recorded in Table 2* 17

Fig. 3 . Root rot development 10 days after inoculating virus-infected and virus-free corn plants with P. grami­ nicola. A = untreated plants; B = inoculated with MDMV C = inoculated with P. graminicola; D = inoculated with %0» MDMV and P. graminicola. 18

Table 2. Root rot indices^ in virus-infected and virus- free corn plants inoculated with P. graminicola at varying intervals.

P. graminicola inoculations at days befpre (-) and after (+) virus inoculation

Treatment -8 0 +1 +3 +4

Untreated 0.0 0.0 0.0 0.0 . 0.0 0.0 With MDMV 0.0 0.0 0.0 0.0 0.0 0.0

With P. graminicola 2.6 1.5 2.5 2.8 2.4 2.5 . With MDMV and P. graminicola 2.7 2.9 3.3 3.9 3.3 3.3

aEach index was a mean of 6 replicates of 6 plants.

LSD at 0.05 = 0.4; at 0 .01 = 0.54. - -

—. The effect of temperature on - development of Pythium ro'o't'~rot in virus-infected and virus- free' corn""plants

Pythium root rot has been reported to be more severe in cold wet soils (1 *7 ). It was of interest to investigate if temperature affects root rot in virus-infected plants.

For this test, plants were kept at relatively low temper­ ature (65°-75°F) and others at a higher temperature (80°- 90°F). Root rot indices were determined 10 and 15 days after inoculating with P. graminicola (Table 3). 19

Table 3. Effect of temperature on root rot development caused by P. graminicola in virus-infected and virus-free corn plants.

Root rot index3.

Days after Temperature inoculation (°p) Virus-free Virus-infected

10 65-75 1.4 2.6** 10 80-90 1.6 2.4**

15 65-75 2.5 3.4** 15 80-90 2.0 3.0**

aMean of 36 plants in 6 replicates.

**Signifleant difference between virus-free and virus- infected plants.

LSD for temperature in virus-free plants = 0.46 at 0.05.

LSD for temperature in virus-infected plants = 0.54 at 0 .0 5 .

Hoot rot severity was greater in virus-infected plants than in virus-free plants at both times and temperatures.

By 10 days, temperature evidently did not affect the sever­ ity of root rot either in virus-free or virus-infected plants. At the end of 15 days, however, root rot of virus- free plants was more severe at the lower temperature, but there was no difference in the root rot between the high and low temperatures in the virus-infected plants. These experiments were repeated with similar results. 20

Data in Table 3 indicate that 15 days after inocula­ tion the virus-free plants kept at relatively low temper­ ature developed more root rot than those at a higher tem­ perature. This could be due to relatively more rapid growth of the host at higher temperature resulting in a lower percentage of the roots with lesions. But in the virus-infected plants the effect of temperature may have been complexed with the effect of virus on the suscepti­ bility of the host. As MDMV increased the susceptibility of corn plants to root rots, the plants at higher temper­ ature still resulted in greater root rot than in virus- free plants.

The effect of inoculum level of P. graminicola in inducing root' rot in' virus -irifec’ted and virus- free corn plants'

In the field, the inoculum level of P. graminicola is relatively low compared to that used in these experi­ ments and root rots usually develop much later. In places where inoculum is high seedling damping-off or severe wilt­ ing usually occurs before the second or third leaf stage. 'mm'K In previous experiments, the normal amount of P. graminicola inoculated to plants in each pot was 1/4 of a 6-day-old

PDA plate culture. To study the effect of fungus inoculum level on the fungus-virus interaction, amounts half the normal and double the normal amounts were used. 21

Fig. 4. The effect of increased inoculum (1/2 plate/pot) of P. graminicola on virus-infected and virus-free c o m plants'? From left to right: First row = inoculated with MDMV and P. graminicola^ second row = inoculated with P. graminicolaj third 'row = inoculated with MDMVj fourth row = uninoculated. 22

Results indicated that when 1/8 of the plate was used, root rot development was slow and did not become severe; none of the plants showed wilting. After 20 days, the root index was 0.4 in the virus-free and 1.4 in the virus- infected plants. With the normal amount of _P. graminicola (1/4 plate) the root rot index was 3.2 for virus-free and 3.8 for virus-infected plants. At double the normal amount of inoculum (1/2 plate/pot), all fungus-inoculated plants started to wilt at a very early stage but the virus-infected plants were more severely affected. As time -progressed, more and more plants were affected and the wilting became irreversible. This is summarized by the data in Table 4. The progress of wilt was very rapid in the first few days in the virus-infected plants. Over 80 percent of the virus- infected plants were affected in the first 10 days after inoculation with P. graminicola, but only about 8 percent of the virus-free plants. All the control plants averaged 0.0 to 0.1. Similar results were obtained from .a repeated experiment. 23

Table 4. Percentage of wilted c o m plants when inoculum level of P. graminicola was doubled (1/2 plate/pot).

Treatments

Days after inoculation with P. graminicola Virus-free Virus -infected

8 1.9 44.1 10 7.7 81.4 12 11.5 81.4 14 23.1 83.1

16 28.5 83.6

20 32.7 84.7

The rate of root rot development in virus -infected and virus -'free plants at normal level Inoculum of P, graminicola' In the previous experiment the data indicated that the virus-infected plants were more rapidly wilted than the plants without virus. By following root rot develop­ ment in similar experiments, it was possible to find the same relationship with root rot development in virus- infected and virus-free plants as was found for wilt de­ velopment. For this test a large number of plants were inoculated with MDMV and P. graminicola as described under

Materials and Methods. Then at intervals of 3 'days, 30-36 plants were randomly collected from each treatment and examined for root rot development. The first two collections 2 k

were made on the second and third day after fungus inocula­ tion ; the remaining were made at intervals of 3 days. The data in Figure 5 indicates that root rot developed

more rapidly in virus-infected than in virus-free plants. A mean root rot index of 3.0 was reached 6 days after in­ oculation of P. graminicola in virus-infected plants, whereas, in the virus-free plants a mean root rot index of 2.7 was reached 15 days after inoculating with P_. grami­ nicola. The control plants averaged 0.0 and thus do. not

appear in the graph. Similar results were obtained from repeated experiments.

Keeping the virus-infected and virus-free plants which were inoculated with P. graminicola beyond the 12th and l4th

day after inoculation resulted in most of the wilted plants . recovering but remaining severely stunted. New adventitious roots, with relatively fewer lesions than old roots, were

being formed, but with relatively more in the virus-free ‘ plants. Pig. 5. The rate of development of P. graminicola root graminicola ofP.developmentrateof The 5.Pig. rot in virus-infected and virus-free corn plants. corn in andvirus-free rot virus-infected

Root rot index 1 0 2 3 3 Days after inoculation Daysafter 6 9 4 O - 12 ■o Virus-free 4 Virus-infected

26

The effect of 3A virus on the susceptibility of corn and wEeat to root rot caused by !P.' gram'i- nicola

As MDMV caused increased susceptibility in young corn plants to root rot caused by P. graminicola and other root rot pathogens, it was considered of interest to investigate if this was a common phenomenon among different viruses in different hosts. To test this hypothesis, the 3A virus, which was mechanically transmissible to both corn and wheat, was used. The plants were inoculated with 3A. virus and then with P. graminicola as described under Materials and Methods. The root rot index was determined 9 a-nd 10 days, in corn and wheat respectively, after inoculating with P. graminicola.

Table 5. The effect of 3A virus on the susceptibility of corn and wheat to Pythium root rot.

. Root rot index3.

Host Virus-free Virus-infected

Corn 3.2 3.7** Wheat 2.4 3 .0**

aEach index was a mean.of 72 corn plants in 12 rep­ licates and 112 wheat plants in 14 replicates.

**Significant at 0.01 level. 27

The results (Table 5) indicated significantly greater root rot in 3A virus-infected plants than virus-free plants both in corn and wheat. The differences, however, between the treatments in this test using corn as a host were not as great as the differences between similar treatments using MDW. The 3A virus does not affect corn as severly as MDMV and this may explain the smaller differences in the severity of root rot between virus-infected and virus- free plants in the latter tests. The results of repeated tests were in the same range as those in Table 5. Corn and wheat plants infected with viruses become more susceptible to root rot diseases caused by fungi. From this, it appears that the virus effect on the plant is not specific with regard to the virus itself and host but affects the susceptibility of the plant in a general way. This may explain why species of Penicillium and Fusarium caused blight in relatively older corn plants that are not usually susceptible.

The effect of MDMV- on the susceptibility of corn plants to seedling blights caused by 'various organisms'

In the first screening test for the pathogenicity of the several fungi, it was found that three species of Pen­ icillium had little effect as root rot pathogens but it was noted that the shoots of the young c o m plants were blighted. Seedling blight of corn usually affects the young germin­ ating plants in the first to third leaf stage, rarely later. 28

But since the virus-infected plants were more susceptible than virus-free plants to root rots, there was a possibil­ ity that (1 ) virus-infected plants would be susceptible to pathogens which do not usually cause blight damage; (2 ) older plants which are usually not affected by seedling blight organisms might also be affected. For these inves­ tigations, plants were inoculated with MDMV and with differ­ ent pathogens. As P. graminicola had been observed to

cause wilting in addition to root rot, it and B. stewartii, which caused wilt in a preliminary test, were included.

Golden Bantam variety of sweet c o m was inoculated with the bacterium using the syringe method and the clip method described under Materials and Methods. Each plant was examined for blight or wilt symptoms

in the young leaves 6-8 days after inoculation. When one or more leaves were affected the plant was counted as blighted or wilted (Fig. 6 ). The differences in the per­ centage of blighted or wilted plants between virus-infected

and virus-free plants were quite striking (Fig. 6 ). More than 40 percent of the virus-infected plants were affected when inoculated with PeniciIlium funiculosum, P. oxalicum, P. purpurogenum, Diplodia zeae, and Pythium graminicola but only 15 to 20 percent of the virus-free plants. Fifteen percent and 13 percent of the virus-infected plants were affected by B. stewartii and F. monilifome respectively but none of the virus-free. F. roseum caused 33 percent 29

Virus-infected Virus-free

•H CD os | | | •H cti -P CD S CD CQ O P O U N O CO O ’H Q) O ctf f t O rH iH fsO *H K •H 3 0(3 O C CD ft H O « U -H +3 •H • *H O 3 £ CQ S ft g ft 3 O 3 . fn JL, • S ft ft 2 hO PQ ft • • • ft ft • ft ft Pig. 6 . Percentage of seedling blight due to different pathogens in virus-infected and virus-free corn plants. 30 blight in the virus-infected but only 3 percent in virus- free plants. Results of the repeated experiments were in the same range and all controls averaged 0.0. The above data indicated that MDMV-infected plants were more susceptible to seedling blight or wilt caused by different pathogens. It was also demonstrated that MDMV-infected plants were affected by seedling blight beyond the stage of usual susceptibility; also that the effect of the virus on susceptibility of plants to differ­ ent pathogens was not confined to a specific pathogen. Further evidence of this general effect of the viruses on the susceptibility of the corn plant to other disorders was obtained in stalk rot experiments.

The effect of MDMV on the sus­ ceptibility of corn plants to stalk rot

Corn plants were planted in large plastic pots so that they would grow to mature size for stalk rot studies. Half of the plants were inoculated with MDMV 8 days after planting and then 'left to grow until 7-8 weeks later. At this time they were inoculated with the stalk rot fungi5

D. zeae^ ?. moniliformej and F. roseum. The internode below the ear shank was inoculated by the cork borer method . described by Williams and Menon (2^!-). A sterile agar disc was inserted into control plants. 31

Table 6 . Stalk rot indices3, in virus-infected and virus- free c o m plants 12 days after inoculation.

Treatment

Pathogen Virus-free Virus-infected

D. zeae 1.3 2 .0** P. moniliforme 1.2 1.7**

P. roseum 1-3 1 .6*

aEach index was a mean of 30-36 plants in 5-6 repli­ cates; indices for control plants ranged between 0.5 and 0.6.

*Significant at 0.05; **significant at 0.01.

The extent of spread of stalk rot was measured on the scale described under Materials and Methods. Significant •

differences in the stalk rot development between the virus- infected and virus-free plants were observed in all the tests (Table 6 ) with the three fungi. In the test with

D. zeae, eight virus-infected plants had fallen to the ground, breaking at the inoculation point. Two plants fell in a similar treatment with P. moniliforme. These

experiments, except the one with D. zeae, were not repeated. 32

Fig. 7. Seedling blight caused by P. purpurogenum in virus- infected (D) and virus-free (C) c o m seedlings. 'K and B are uninoculated and MDMV-inoculated controls, respectively. DISCUSSION

Results from this investigation indicate that young corn and wheat plants infected with MDMV or 3A virus become more susceptible to root rots, stalk rots, and seedling blights, caused by several different pathogens. Data on root rots also indicated that the virus-infected plants become susceptible to organisms that may be only secondary or weak pathogens and usually not seriously virulent to young virus-free corn plants. This was indicated in the tests in which the virus-infected and the virus-free plants were inoculated with such fungi as Fusarium oxysporum, F. moniliforme, F. roseum, Helminthosporium pedicellatum, and Diplodia zeae, which usually are not serious root rot patho­ gens of young corn plants. In tests with _P. graminicola, l,

the data indicated that the root rot index of virus-infected plants was in certain cases more than twice the root rot index of virus-free plants. Some fungi, however, originally isolated from corn roots in the field were not pathogenic to either virus-infected or virus-free corn seedlings.

Increase of susceptibility in plants infected with viruses may be a general phenomenon rather than a specific virus in a specific host. For in this research, it was

demonstrated that two different viruses increased suscep­ tibility of corn to several root rot, stalk rot, and

33 34 seedling blight organisms and one in wheat increased sus­ ceptibility to Pythium root rot. Viruses may increase sus­ ceptibility as the result of a general weakening of host plants, but it could also be due to effects on specific processes. Millikan and Pickett (11,12) found less DNA, RNA, carbohydrates, and zinc in apple leaves inoculated with stem pitting virus than in healthy leaves. These observations indicate that physiological effects of virus infection in apple might be long lasting and be a predis­ posing factor in the susceptibility of the host to subse­ quent infection with other pathogens. This may very well be the case with the effect of viruses on the susceptibility of corn and wheat to disorders caused by other pathogens.

Susceptibility of corn root rot and stalk rot patho­ gens has been linked with the mineral nutrition of the host. Thayer and Williams (17) found that an increase in the concentration of phosphorus- in gravel culture solutions reduced the development of both root and stalk rot of corn.

Increase in nitrogen, increased amount of stalk rot when potassium concentration was at medium and high level, but had no effect at low level. These findings elucidated the importance of balanced mineral supply to the plant. Vanterpool (19) concluded that improper balance of available phosphorus and nitrate nitrogen led to unbalanced metabolism of wheat seedlings and predisposed them to fungal attack. MDMV or 3A virus-infected c o m plants may be altered similar 35

to that of excesses or deficiencies of certain elements. It seems reasonable to infer that a virus-infected plant . would need a larger supply of certain elements used in the synthesis of the virus, either in contributing to the structural components of the virus or in functioning as part of enzymes. The deficit created in the host would 'result in changed metabolism, a process that may lead to increased susceptibility of the host to other pathogens.

In the field, the phenomenon of increased suscepti­ bility in virus-infected plants would result in greater

losses of the crop as a result of increased susceptibility . of the host. For fungi and other organisms which are usually not pathogenic to c o m or wheat plants would be

effective in causing some losses. Diseases like seedling

blight of corn would become common in older plants which are usually not susceptible beyond the second or third leaf stage. This was confirmed in this research in which a significantly higher percentage of virus-infected plants were blighted by pathogens which had had little or no effect on virus-free plants. SUMMARY

C o m and wheat plants growing in a greenhouse were used to test the effect or virus infections on the susceptibility of plants to other pathogens. Two viruses, maize dwarf mosaic, and 3A, were used. Several pathogens, one bacterial and several fungal, were used in testing the susceptibility of the plants. In tests in which Pythium graminicola was used as a root rot causing agent, it was found that MDMV- infected plants developed more severe root rot than virus- free plants. Whereas the virus-free plants in a relatively lower temperature (65°-75°F) developed a significantly more severe root rot than the plants at higher temperature (80°-90°F), the root rot indices of virus-infected plants in the lower and higher temperatures did not differ signif­ icantly. This may be due to the rapid increase of virus at relatively higher temperature. The increase of virus created a greater effect on the susceptibility of the plant at the temperature than at lower temperature at which the virus increase may not be so rapid. When 3A virus was used in corn and wheat plants, root rot development was greater in virus-infected plants.

Diplodla zeae and Fusarium moniliforme caused more root rot in the MDMV--infected than in virus-free plants.

36 37

A few fungi tested were not pathogenic to either virus- infected or virus-free plants.

Wilting of corn plants due to P. graminicola was over So percent in virus-infected plants compared to less than 33 percent in virus-free plants. Bacterium stewartii also caused wilt but only in virus-infected c o m plants. D. zeae, P. roseum, Penicillium funiculosum, P. oxalicum, and P. purpurogenum caused seedling blight which was greater in virus-infected plants than in virus-free plants. In case of D. zeae and F. moniliforme the blight was only in plants with virus.

The effect of viruses in increasing the susceptibility of corn plants was also noted in stalk rot studies. When

D. zeae, F. moniliforme, and F. roseum were used as c o m stalk rot pathogens, relatively more stalk rot was recorded in virus-infected plants. LITERATURE/ CITED

1. Carpenter, C. W. 1934. Predisposing factors in Pythium root rot. Hawaiian Planters Record 38:279-338. 2. Castano, J. J. and M. K. Kernkamp. 1956. The influence of certain plant nutrients on infection of soy­ beans by Rhizoctonia solani. Phytopathology 46:326-328.

3. Farley, J. D. and J. L. Lockwood. 1964. Increased sus­ ceptibility to root rot in virus-infected peas. (Abstr.) Phytopathology 54:892.

4. Farley, J. D. and J. L. Lockwood. 1964. Increased sus­ ceptibility to root rot in virus-infected peas. Phytopathology 54:1279-1280. - -

5. Foley, D. C. 1955. The effect of fertility on stalk rot of corn in Pennsylvania. Ph.D. thesis, The Pennsylvania State University, 6. Forrest, W. D., S. D. Hensely, J. D. Candia and T. P. Pirone. 1964. An abnormality resembling corn stunt virus in Louisiana. Plant Disease Reptr. 48:645-646.

7. Hoppe,- F~.~ E. 1949. Differences in Pythium injury to corn seedlings at high and low soil temperatures. Phytopathology 39:77-84. 8. Lockwood, J. L. and L. E. Williams. 1957. Inoculation and rating methods for bacterial wilt of sweet corn. Phytopathology 47:83-87.

9. Martin, J. P. 1934. Symptoms of malnutrition manifested by sugar cane plant when grown in culture solu­ tions from which certain essential elements are omitted. Hawaiian Planters Record 38:3-31.

10. McNexf, G. L. and E. L. Spencer. 1939. Effect of nitrogen supply of sweet corn on the wilt bacter­ ium. Phytopathology 29:1051-1067.

11. Millikan, D. F. and E. E. Pickett. 1964. Biochemical patterns in leaf tissue from virus-infected and disease-free apple. I. Nucleic acid constituents. Proc. Amer. Soc. Hort. Sci. 85:48-52. 38 39

12. Millikan, D. F. and E. E. Pickett. 1964. Biochemical patterns in leaf tissue from virus-infected and disease-free apple. II. Some cation effects. Phytopathologische Zeitschrift 50:89-91. 13. Otto, H. J. and H. L. Everett. 1956. Influence of nitrogen and potassium fertilization on the in­ cidence of stalk rot of corn. Agronomy Jour. 48:301-305. 14. Schmitthenner, A. F. and J. ¥. Hilty. 1962 . A method for studying post emergence seedling root rot. Phytopathology 52:177-179. 15. Smith, H. C. 1962. Is barley yellow dwarf mosaic virus a predisposing factor in the common root rot disease of wheat in Canada? Can. Plant Disease Survey 42:143-148.

16 . Spencer, E. L. and G. L. Me New. 1938. The influence of mineral nutrition on the reaction for studying root rot pathogens. (Abstr.) Phytopathology 47:30. 17. Thayer, P. L. and L. E. Williams, i9 6 0 . Effect of nitrogen,, phosphorus, and potassium concentra­ tions on the development of Gibberella stalk rot and root rot of corn. Phytopathology 50:212-214.

18. Thomas, E. S. and D. H. Bowman. 1953. The rusts and other diseases of small grains in Mississippi. Plant Disease Reptr. 37:142-147.

19. Vanterpool, T. C. 1935. Studies on browning root rot of . III. Phosphorus-nitrogen relations, of infested fields. IV. Effects of fertilizer amendments. V. Preliminary plant analyses. Can. Jour. Res. 13:220-250. 20. Walker, J. C. and R. E. Foster. 1946. Plant nutrition in relation to disease development. III. Fusarium wilt of tomato. Am. Jour. Bot. 33:259-264. 21. Watson, R. D. 1964. Virus-fungus relationship in a root rot complex in red clover. (Abstr.) Phyto­ pathology 54:911. 4o

22. Williams, L. E. and L. J. Alexander. 1965. Maize dwarf mosaic, a new corn disease. Phytopathology 55:802-804.

23. Williams, L. E., L. J. Alexander, and H. A. Runnels. 196 5. A virus isolated from red-streaked corn grain. (Abstr.) Phytopathology 55:1083-1084. 24. Williams, L. E. and S. K. Menon. 1957. A cork borer • technique of inoculating c o m plants with stalk rot fungi. Plant Disease Reptr. 4l:111-114. AUTOBIOGRAPHY

I, Nelson Peter Mwanza, was born in Malawi on May 13, '

1937- I received my secondary education at Zomba Secondary School, Malawi and at Munali Secondary School, Lusaka, Zambia. I received my undergraduate training at The University College of Rhodesia and Nyasaland, Salisbury, Rhodesia, which granted me the Bachelor of Science degree in 1961. After 10 months with the Malawi Government Department of Agriculture as a technical officer, I was awarded a Ford International Fellowship to study at The Ohio State University which granted me the Master of

Science degree in 1964. In 1963* I was appointed a teaching assistant in the Department of Botany and Plant Pathology at The Ohio State University. I have held this position until the present time while completing the requirements for the Doctor of Philosophy degree.

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