FACTORS AFFECTING THE PRODUCTION OF LOCAL LESIONS BY BEAN COMMON MOSAIC VLRUS. (BC-MV) Thesis: for the Degree of M. S. MICHLGAN STATE UNIVERSITY GUSTAVO ENRIQUE TRUJILLO 1971 . -‘*‘§HO‘-"‘4 LIBRARY MiChigan State University ILL. I]. 8 r HDAG & .snnx swam mc. H #69533 3 AA OO3 ABSTRACT FACTORS AFFECTING THE PRODUCTION OF LOCAL LESIONS BY BEAN COMMON MOSAIC VIRUS (BCMV) BY Gustavo Enrique Trujillo All attempts to consistently obtain local lesions on rub-inoculated leaves of bean as reported by previous workers were unsuccessful. However, local necrotic lesions were consistently obtained on primary leaves of the Monroe bean variety rubbed with the type (V1), New York 15 (V15), and 123 (V123) strains of bean common mosaic virus (BCMV). Under greenhouse temperature (22-27 C) and light (natural) conditions, necrotic lesions about 1 mm in diameter were initially observed and could be counted 4-5 days after inoculation. Similar lesions were formed on rub-inoculated trifoliolate leaves of Monroe bean. Inoculation of leaves with the rough end of a pestle was superior to other inoculation devices such as cheesecloth, foam rubber pads, and "Q-tips." The number of local lesions obtained with V and l V15 on primary leaves of Monroe bean was inversely related to dilution of inoculum, and was relatively uniform among Gustavo Enrique Trujillo the four half leaves on a plant. Uniform lesion counts were obtained when inocula were prepared in 0.01 Molar NaZHPO -KH PO buffers of pH 6.4-7.8. 4 2 4 Temperature affected the quality, as well as the quantity of BCMV-induced local lesions on Monroe bean. Lesion number and quality (shape and size) were highest at 20 C, good at 24 C, and poor at 28 C and 16 C. Preinocu- lation shading of plants for 24 hr increased the number of local lesions formed; lesions on such plants were somewhat larger than lesions on unshaded plants. The Optimum age of plants for primary leaf inocu- lation, as measured by local lesion production, was 9-12 days after planting; lO-days-old plants developed the largest number of local lesions. Approved: Ma 'oig Professor Department Chairman FACTORS AFFECTING THE PRODUCTION OF LOCAL LESIONS BY BEAN COMMON MOSAIC VIRUS (BCMV) BY Gustavo Enrique Trujillo A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1971 ACKNOWLEDGMENTS The author wishes to express his sincere apprecia- tion to Dr. A. W. Saettler, Research Plant Pathologist, United States Department of Agriculture, and Assistant Professor, Department of Botany and Plant Pathology, who provided counsel, guidance, and encouragement throughout the entire investigation, and during the preparation of this manuscript. Appreciation is likewise extended to Drs. W. J. Hooker and H. H. Murakishi, who served as members of my guidance committee, and who reviewed this manuscript. The Fondo National de Investigaciones AgrOpecuarias (Venezuela) provided financial assistance to the author during this graduate study program. ii TABLE OF CONTENTS ACMOWLEDGMENTS O O O O O I O O O O O O O O O I O 0 LIST OF TABLES O O O O O O O O O O O O O O O O O 0 LIST OF FIGURES O O O O O O O O O O O O I O O O O 0 Chapter I. II. INTRODUCTION 0 O O O O O O O O O O O O O 0 Historical Background . . . . . . . . . . Symptoms . . . . . . . . . . . . Physico-Chemical Properties . Modes of Transmission . . . . Seed Transmission . . . . Mechanical Transmission . . Insect Transmission . . . Host Range . . . . . . . . BCMV Strains . . . . . . . . . . . Significance and Utility of BCMV Local Lesion Host . . . . . . . . . . . Previously Reported Examples of BCMV-— Induced Local Lesions . . . . . . . . Factors Affecting Local Lesion Assay Light . . . . . . . . . . . . . . . Temperature . . . . . . . . . . . . Age of Plant . . . . . . . . . . . Methods of Virus Preparation and Inoculation . . . . . . . . . . . . . . . Different Rubbing Methods . . . . . . . Additives that Increase Infection Efficiency . . . . . . . . . . . . Objectives of This Study . . . . . . . . we. MATERIALS AND METHODS . . . . . . . . . . . Plant Material . . . . . . . . . . . . . Virus Material . . . . . . . . . . . . . Inoculation Procedures . . . . . . . . . iii Page ii vii mnppppwww H KC 12 12 13 14 15 15 15 16 17 17 18 Chapter III. RE IV. DI Data Collection . . . . . . . . . . . . . Experiments . . . . . . . . . . . . . . . Preliminary Attempts to Reproduce Lesions Reported by Zaumeyer and Goth . Best Method of Inoculation . . . . . . Inoculum pH . . . . . . . . . . . . . . Relationship Between Dilution of Inoculum and Lesion Formation . . . . . Relationship Between Age of Leaf and Lesion Formation . . . . . . . . . . . Pre- and Post-Inoculation Light Treatment . . . . . . . . . . . . . . . The Effect of Temperature . . . . . . . SULTS I O O O O O O O O O O O I O O O O 0 Preliminary Attempts to Reproduce Lesions Reported by Zaumeyer and Goth . . . . . . Best Method of Inoculation . . . . . . . Inoculum pH . . . . . . . . . . . . . . . Relationship Between Dilution of Inoculum and Lesion Formation . . . . . . Relationship Between Age of Leaf and Lesion Formation . . . . . . . . . . . . Pre- and Post-Inoculation Light Treatment . . . . . . . . . . . . . . . . Effect of Temperature on Lesion Formation . . . . . . . . . . . . . . . . Thermal Inactivation of BCMV . . . Local Lesion Formation by BCMV on Trifoliolate Leaves of Monroe Bean . . . Reaction of Monroe Bean to Other Plant Viruses . . . . . . . . . . . . . . . . . SCUSSION O O O O O O O I O O O O O O O O V 0 SUMMARY 0 O O O O O I O O O O O O O O O O 0 REFERENCES iv Page 19 20 20 20 21 21 22 22 23 25 25 31 33 34 35 43 46 52 52 56 57 66 68 Table 1. 10. LIST OF TABLES Reported strains of BCMV (bean common mosaic ViruS) O O O O O O O O I O O O O O O O O 0 Reactions of different bean varieties to strains of BCMV (bean common mosaic virus) The effect of constant air temperature (twelve hour photo period) on symptom response of primary bean leaves inoculated with 3 strains of bean common mosaic virus (BCMV) . . . . . . . . . . . . . . . . . . The effect of constant air temperature (con- tinuous light) on symptom response of primary bean leaves inoculated with 3 strains of bean common mosaic virus (BCMV) Symptom response of primary bean leaves inoculated with 3 strains of bean common mosaic virus (BCMV) under greenhouse conditions (winter season) . . . . . . . . The effect of inoculation method on local lesion formation by BCMVl on primary leaves of Monroe bean . . . . . . . . . . . The effect of inoculation method on local lesion formation by BCMVl on primary leaves of Monroe bean . . . . . . . . . . . The effect of inoculum pH on local lesion formation by BCMVl on primary leaves of Monroe bean . . . . . . . . . . . . . . . . The effect of inoculum pH on local lesion formation by BCMVl on primary leaves of Monroe bean . . . . . . . . . . . . . . . . The effect of inoculum dilution on local lesion formation by BCMVl and BCMV15 on primary leaves of Monroe bean . . . . . . . Page 27 28 29 32 32 33 34 35 Table Page 11. The effect of inoculum dilution on local lesion formation by BCMVl and BCMV15 on primary leaves of Monroe bean . . . . . . . . 35 12. The effect of inoculum dilution on formation of local lesions by BCMVl on primary leaves of Monroe bean plants of different ages . . . . . . . . . . . . . . . . . . . . 36 13. The effect of different light treatments on local lesion formation by BCMVl on primary leaves of Monroe bean . . . . . . . . . . . . 46 14. The effects of different lengths of pre- inoculation shading on local lesion for- mation by BCMVl on primary leaves of Monroe bean . . . . . . . . . . . . . . . . . 50 15. The effect of constant air temperature on local lesion formation by BCMV on primary leaves of Monroe bean . . . . . . . . . . . . 52 16. Local lesion production by BCMV1 on trifolio- late leaves of Monroe bean inoculated 25 days after planting . . . . . . . . . . . . . 53 vi LIST OF FIGURES Figure Page 1A. Typical symptoms of bean common mosaic virus (BCMV) on trifoliolate leaves of Rainy River I O O O O O O O O O O O O O O C O O O 3 1B. Plant of Rainy River bean showing systemic invas ion Of BCMV O O O I O O O C O C O O O O 3 2. Symptom response of bean leaves inoculated with BCMV at 20 C. (A) Vein necrosis (VN) on Great Northern UI-31 inoculated with BCMV15 (B) Ghost Spots (GS) on primary leaves of Great Northern UI-123 inoculated with BCMVl (arrow) . . . . . . . . . . . . . 26 3. Two types of local lesions on primary leaves of Monroe bean inoculated with BCMV (A) Local necrotic lesion on primary leaf of Monroe bean (B) Ringspot lesion on primary leaf of Monroe bean . . . . . . . . . . . . . . 30 4. The effect of dilution of inoculum on local lesion production by two strains of BCMV on primary leaves of Monroe bean . . . . . . 37 5. Primary leaves of Monroe bean inoculated with different dilutions of BCMV1. Dilution endpoint of BCMV is between 1:800 and 1:1000 . . . . . . . . . . . . . . 38 6. Seedlings of Monroe bean at various times after planting. From right, 8, 9, 10, ll, 12, and 13 days after planting . . . . . 40 7. The effect of inoculum dilution on formation of local lesions by BCMVl on primary leaves of Monroe bean plants of different ages . . 42 vii Figure 10. 11. 12. 13. 14. The effect of different light treatments on local lesion formation by BCMVl on primary leaves of Monroe bean (A) Greenhouse-Greenhouse (B) Greenhouse-24 hr. dark (C) 24 hr. dark-24 hr. dark (D) 24 hr. dark-Greenhouse . . . . . . . . The effect of different light treatments on size of local lesions produced by BCMVl on primary leaves of Monroe bean . . . . . . Ringspot-type lesion on primary leaf of Monroe bean inoculated with BCMVl at 24 C Severe vein necrosis on primary leaf of Monroe bean inoculated with BCMVl at 28 C. Local lesions are indistinct and difficult to count . . . . . . . . . . . . . . . . . Hypersensitive-type reaction of primary leaf of Topcrop bean inoculated with BCMVl at 32 C O O I O O I O O O O O C O O O O O O 0 Appearance of BCMV-induced lesions on inocu- lated primary leaves of Monroe bean at different times of the year (A) Fall (October) lesions are Ringspot with necrotic center (B) Winter (January) lesions are local necrotic lesions (C) Spring (April) lesions are Ringspot with non-necrotic center. All lesions were formed in the greenhouse . . . . . . . Local lesion production on trifoliolate leaves of Monroe bean inoculated with BCMVl O O O O O O O O O C O O O O O O O 0 viii Page 44 45 47 48 49 51 54 CHAPTER I INTRODUCTION Historical Background Bean common mosaic virus (BCMV, Bean Virus 1, Marmor phaseoli, Holmes) was first reported in Russia by Iwanowski (22) about 1894. Since that time mosaic has been reported in many countries throughout the world (52) and is almost always present wherever susceptible bean varieties are grown. This seed, insect and mechanically transmissible virus can cause large field losses in sus- ceptible bean varieties. In 1946 Zaumeyer (47, 51) reported the disease widespread in several western states; the disease was most severe in Colorado, where the yield in some fields was reduced by about one third. Outbreaks of bean mosaic in 1945 and 1947 in Idaho were severe in the variety Red Mexican 3; 100% plant infection was found in at least one field. Field reductions due to mosaic in fields of this same variety were considerable in western Washington. In 1951 Andersen (1) reported bean common mosaic in cranberry beans growing in Montcalm County, Michigan. Symptoms \\ The symptoms of BCMV may vary according to the variety of bean infected, time of infection, environmental conditions and strain of the virus involved. Usually mottling and various types of leaf malformation are present (Figure l); stunting of plant development is usually evident also. Trifoliolate leaves infected with mosaic usually have irregularly shaped, light yellow and green areas (52, 38). Bean plants infected early in the growing season are more likely to be stunted and yield less than plants infected later. (lPods on severely in- fected plants are undersized and contain fewer ovules than those produced on normal plants (52). Usually, high temperatures favor Optimum symptom development; symptoms tend to be masked at temperatures above 30 C and below 15 C (38) . Physico--Chemical Properties The thermal inactivation point of BCMV lies between 56 and 58 C (52). The dilution end point is 1:1,000 and longevity in_yi£52_between 24 to 32 hours at laboratory temperatures. All of these properties were studied, how- ever, without the benefit of a local lesion host. Accord- ing to Brandes and Quantz (38), BCMV is a typical rod- shaped particle measuring 750 mu in length. Figure 1A. Typical symptoms of bean common mosaic virus (BCMV) on trifoliolate leaves of Rainy River. Figure 1B. Plant of Rainy River bean showing systemic invasion by BCMV. Modes of Transmission Seed Transmission Initial establishment of BCMV in a bean field is usually through the planting of virus-infected seed (27). Incidence of seed transmission as high as 30-50% has been reported (51).;EFajardo (15) showed that incidence of infected seed harvested is correlated with certain stages of plant growth. Mosaic infection which occurred prior to blossom gave the greatest number of infected seeds. Mechanical Transmission .KBCMV is typical of many mosaic viruses in that it is transmitted through mechanical means, including mach— inery, man, and animals. The virus is easily transmitted to healthy plants by rub-inoculating leaves with sap ex- pressed from infected leaves. Insect Transmission Insect transmission of BCMV has been shown for a number of aphid species (28, 50), including the common~ Myzus persicae. Host Range In addition to common bean (Phaseolus vulgaris L.), the following plant species have been found experimentally to be susceptible to BCMV: Species: Cajanus cajan (L) Millsp. (Spring pigeon pea), Canavalia ensiformis (L) D. C. (jack bean), Cicer aretinum L. (chick pea), Crotalaria spectabilis Roth. (crotalaria), Glycine max (L) Merr. (soybean) cv. Black hawk, Lespedeza striata (Thumb.) H. & A. (common lespedeza), Lupinus albus L. (yellow lupine), Phaseolus acutifolius Gray var. latifolious Freeman (tepary bean), P. calcaratus (52). Roxb. (rice bean), P. lunatus L. (lima bean) cv. Henderson, Bush, Thaxter, and Fordhoos 242, P. mungo L. (urd bean), Sesbania exaltata (Raf.) Cory, and Stizolobium deeringianum Bort (velvet bean). In_196l Quantz (29) re- ported Vigna sinensis (Torner) Savi (COWpea) to be system- ically-susceptible to BCMV. In 1964 Zaumeyer and Goth (49) added these additional hosts: Cassia tora, Lens culinaris Medick, and Trifolium subterraneum L. Under natural conditions, BCMV has been isolated from on1y~ Phaseolus vulgaris L. (52). BCMV Strains A number of strains of BCMV have been reported and are summarized in Table 1. In 1939 Burkholder and Richards (31) found plants showing typical common mosaic virus symptoms in two fields of Michelite Navy (pea) beans near Batavia, New York. Because Michelite was reported resis- tant to BCMV (l4), and because other resistant varieties such as Great Northern UI-lS, Red Mexican UI-3, Red Mexican UI-34 and Norida were also susceptible to the new virus, the authors concluded that this was a new strain of BCMV. The same or very similar strain was found in Idaho in 1946 (9) in a field of Great Northern UI-15 beans: this par- ticular strain-of BCMV is commonly called New York 15 (NY 15) or 1943 strain (36). In 1954 (10) another unreported strain of BCMV was discovered in Idaho infecting Great Northern UI-123 beans. Other bean varieties which pos- sessed dominant resistance to the type strain were either resistant or susceptible to the new strain. The varieties Great Northerns UI-l6, UI-31, UI-59, UI—81 and UI-123 are resistant to the type and New York 15 strain, but suscep— tible to the New 1954 strain. This strain has been re- ferred to as 1954 strain, Idaho strain or BCMV 123. Table 1. Reported strains of BCMV (bean common mosaic virus). Year When Strain Name Described Workers Type (V1) 1894 Iwanowski New York 15 (V15) 1943 Richards and Burkholder 123 (V123) 1959 Dean and Wilson- Western 1961 Skotland and Burke Florida 1964 Zaumeyer and Goth Mexican 1969 Silbernagel In 1964 Zaumeyer and Goth (49) isolated an addi- tional unreported strain from an experimental pole bean line, seed of which was received from R. A. Conover of Florida. Varieties resistant to the type and N.Y. 15 strains were also resistant to the new strain; it was mainly on the basis of severity of mosaic symptoms that the new strain could be distinguished. Several COWpea varieties were also useful in distinguishing the new strain. Because of the host from which it was first isolated, it is called the Florida strain. A virus related to BCMV was reported in 1961 (37) from the Pacific Northwest. In addition to common bean, the "new" virus infected lima bean, COWpea var. Ramshorn, chick pea, Tepary bean, and spinach. T0pcrop, Sanilac, Idaho Bountiful and Michelite were all immune. The virus was inactivated in 10 minutes between 60-65 C; possessed a dilution end point between 1:10,000 and 1:50,000, and was termed the Western Bean Mosaic Virus (WBMV). More recently, Silbernagel (36) isolated a virus from the bean line P.I. 1976903', which he named the Mexican strain of BCMV. The bean variety Red Mexican UI-35 is susceptible and the variety Improved Tendergreen is resistant to the Mexican strain, distinguishing it from all of the other reported strains. Silbernagel used cross protection tests to show relationships with the NY-15 and Florida strains. There are additional reports of other viruses re- lated to BCMV (13, 16). In 1960 (46) Yerkes and Patino reported the severe strain of BCMV from Mexico but Grogan and Kimble (1?) later showed that this strain was actually related to southern bean mosaic virus. Snow (39) isolated a virus from gladiolus that was capable of attacking plants possessing the dominant type of resistance derived from Corbett Refugee. Later, however, other workers (49) presented evidence that the gladiolus virus was a strain of yellow mosaic virus. All of the reported strains of BCMV are differentiated on the basis of their reactions on different bean varieties as shown in Table 2. Table 2. Reactions of different bean varieties to strains of BCMV (bean common mosaic virus).a BCMV Strain Bean Variety V1 V15 V123 Florida Mexican Western Bountiful S S S S S S Great Northern UI-3l . R R S R S R Great Northern UI-123 R R S R S S Pinto UI-lll R S R R R R Sanilac R S R R R R Topcrop R R R R R R a S = susceptible = resistant Significance and Utility of a BCMV Local Lesion Host Although BCMV has been recognized as a serious bean disease for a long time, its purification has only been partial primarily because no local lesion host is available to estimate virus activity during the purifi- cation process. Quantitative assay using a local lesion host would allow detailed study of the physical and chemical prOper- ties of BCMV and its strains, eg. thermal stability, sedi- mentation constant, isoelectric point, longevity in_!itgg, resistance to certain enzymes and other chemicals. A local lesion host for BCMV would also be useful in cross protection tests to study relationships between the dif— ferent strains and other viruses. The factors which in- fluence the establishment and multiplication of the virus in a host could also be examined. These are just several of the reasons why a number of workers (35, 40, 48, 49) have attempted to find a local lesion host for BCMV. Previously;Reported Examples of BCMV-- Induced Local Lesions Using high temperatures (32 C) after inoculation, Thomas and Fisher (40) showed that BCMV produced necrosis and occasionally, necrotic lesions on the rubbed leaves of bean varieties such as Idaho Refugee and Topcrop. Only 10 bean varieties which possess the dominant type of mosaic resistance derived from Corbett Refugee give this reaction. The virus later becomes systemic, killing the terminal growth and often killing the plant (18). Although it is a rapid test to detect dominant types of resistance to BCMV, high temperature (32 C) is necessary and the local necrotic lesions which do form are not produced consistently. Quantz in 1957 (29) described faded brown rings and vein necrosis on certain dry bean varieties when rub-inoculated with BCMV. However, these ringed lesions were not formed in reproducible numbers on inoculated leaves, and the lesions required a long period for appearance (3 weeks). Quantz subsequently reported the production of discrete, brown necrotic lesions by BCMV on detached leaves of T0pcr0p in closed petri dishes at 32 C. In 1962 Schneider and Worley (35) reported a similar type of lesion which formed on detached leaves of Tender Long 15, as well as Topcrop beans inoculated with BCMV type or New York 15 strain. Detached leaves were placed in_a nutrient medium incubated in the dark at 32—35 C. Various factors influenced the number and character of the lesions pro- duced: inoculum concentration, preinoculation or post- inoculation exposure to high temperature and presence or absence of glucose in the medium. The nature and appear- ance of these lesions on TopcrOp suggest that they are a modified hypersensitive reaction of the type initially described by Thomas and Fisher. 11 Zaumeyer and Goth (48, 49) have reported two types of local lesions. The first type was produced by the type, New York 15 and Florida strains of BCMV on leaves of Stringless Green Refugee, Plentiful, Potomac and Pinto UI-lll. The lesions appeared 4 days after inoculation under normal greenhouse conditions, and also in a 27 C growth chamber under continuous light. Because these lesions were not consistently observed, they suggested that certain-unknown environmental conditions were nec- essary for their formation. The second type of local lesion produced by BCMV and two of its strains was a brown, ring-like lesion 5-7 mm in diameter which appeared 3-4 days after inoculation of leaves of an unnamed pole bean cross (obtained from R. A. Conover of Florida). Later, however, the virus became systemic causing typical mottling of the upper leaves. Efforts of A. W. Saettler to obtain seed of this unnamed cross were unsuccessful, and, according to R. A. Conover (personal correspondence) "none of these stocks are available now." Dr. Conover, did, however send Dr. Saettler seed of the Dade bean variety which he indicated is almost certainly a sister line of the one used by Zaumeyer and "probably should react as did the stock Bill (Zaumeyer) used." 12 In 1969 (36) Silbernagel reported a hypersensitive reaction of Mexican BCMV strain on leaves of Topcrop plants maintained at 32 C after inoculation. This hyper- sensitive reaction of Topcrop is a characteristic for all strains of BCMV. A. Andersen observed some years ago that Monroe, a Navy (pea) bean variety released by Cornell University, produced well-defined necrotic lesions when inoculated with N.Y. 15 strain (unpublished information). Andersen bulked a large numbbr of single plant selections from plants showing the most ideal lesion development, but did not study the lesions further. Factors Affecting_Local Lesion Assay Although many factors affect local lesion produc- tion qualitatively and quantitatively, light and tempera- ture have been studied most extensively. Light " Bawden and Roberts (4) reported that shading plants for 24 hours prior to inoculation increased susceptibility to infection with tobacco necrosis, tomato bushy stunt, tobacco mosaic and tomato aucuba viruses on tobacco var. White Burley, Nicotiana glutinosa, tomato var. Kondine Red and tobacco and tomato, respectively. 13 Desjardins (12) working with alfalfa mosaic virus on beans, reported that lesions were larger than normal if the leaves were kept in the dark or shaded immediately after inoculation. Huguelet and Hooker (21) using potato virus on Gomphrena globosa reported that a period of darkness after inoculation seemed to suppress lesion formation. Temperature *Bawden (2, 13) stated that the most general effect of increasing temperature is to shorten the interval be- tween infection and the production of lesions. Temperature may affect both the severity and the type of symptom de- pending on the virus-host combination used. Kasanis (24) reported that when Nicotiana glutinosa and bean plants were kept at 35 C for about 24 hrs., their subsequent susceptibility to tobacco mosaic, tobacco necrosis and tomato bushy stunt viruses was increased as measured by local lesions. In 1958 Yarwood (44) reported that lesion counts of TMV were increased 2-3 times when Pinto bean leaves were heated for 30-45 sec. at 50 C, 5-10 hours after inoculation. Ross (32), working with potato virus Y on Physalis floridana reported that postinoculation temperature ex- erted a great effect on local lesion formation. Although 14 more local lesions were produced at 27 C than at 18 C, lesions at 18 C were more distinct and more easily counted than those at 27 C. Ross also found that preinoculation temperature had very little effect on the subsequent pro- duction of lesions on P. floridana. Age of Plant Young, well fertilized, rapidly growing plants are usually the best source of inoculum for virus studies (26, 33). Matthews (26) indicates that the susceptibility of leaves to a particular virus may change rapidly as the leaves age. Bawden (2), working with tobacco necrosis virus on French bean (P. vulgaris) showed that lO-day-old plants gave many infection points, but that 13 to l4-day—old plants gave no infection points. Shein (34) found that susceptibility of beans to TMV, as measured by local lesion numbers, was low at the time of leaf unfolding, maximum when leaves were 20—40% expanded, and low again when leaves were approximately 100% fully expanded. Ross (32), working with potato virus Y on Physalis floridana, found that lesion formation was optimum when plants were inoculated during early flowering. Crowley (8) found with lettuce necrotic yellow virus on Nicotiana glutinosa that the most important 15 factor affecting susceptibility was the age of the plant; young leaves, one-half to three-fourths expanded at the time of inoculation were more susceptible than older leaves. Methods of Virus Preparation and Inoculation Different Rubbing Methods The more common devices used for rub inoculation are: finger, Spatula, cheesecloth pad, pestle and brush. The standard rubbing method is to dip the device into virus suspension, and to rub the virus suspension onto the upper surface of young leaves previously dusted with an abrasive. Leaves are quickly rinsed with water after inoculation. Yarwood (43) indicated that all of these methods if prOperly used will give similar results. Additives that Increase Infection Efficiency It has been shown consistently that efficiency of mechanical inoculation is increased when an abrasive material is used (added to the inoculum or sprinkled over the leaves) (43, 44, 45). yflhe most commonly used abrasives are carborundum (400-500 mesh) or diatomaceus earth such as Celite. In 1952 Yarwood (42) showed that the addition of 1% solution of dipotassium phosphate to the inoculum 16 ~%\ greatly increased virus infectivity, but the way in which phosphate increases the number of successful infections is not yet understood (45). Objectives of This Study The objectives of this investigation were three- fold: l. Attempt to repeat the results of Zaumeyer (49) and others in relation to production of local lesion by BCMV on different bean varieties. 2. To test the utility of Monroe bean as a local lesion host. 3. To determine some of the environmental and physiological factors which influence local lesion forma- tion by BCMV on Monroe. CHAPTER II MATERIAL AND METHODS Plant Material Seed of various bean varieties was planted in a soil mix composed of equal parts (1:1) muck soil and ver— miculite. Plants were contained in 32 oz. wax-lined card- board cartons, 3 plants per carton (Figure 6). Plants were watered alternately as needed with modified Hoagland's solution, tap water, and iron solution (Sequestrene, Fe as metallic 1.8 ppm.). Unless indicated otherwise, all plants were grown under normal greenhouse conditions of alternating air temperature (22-27 C); natural daylight was supplemented 12 hrs. daily during winter season with fluorescent lamps (60 ft-c). Virus Material All of the BCMV strains used in this study were initially established from virus-infected seeds (collected in 1961) or from desiccated plant tissue kept in the freezer since 1961. All of the strains were checked for purity and pathogenicity on the differential bean varieties prior to and during this study. Fresh juice inoculum was 17 18 prepared from Rainy River (BCMV type), from Pinto UI-lll (N.Y. 15 strain), and from Great Northern 123 (BCMV-123). Young trifoliolate leaves showing good mosaic symptoms (about 20-24 days after plant inoculation) were removed and triturated in a sterilized mortar. The macerated tissue was pressed through one layer of cheesecloth and dilutions prepared in 0.01 M phosphate (Na2 HPO4 - KH2 P04) buffer pH 7.4. In some cases, the infected leaves were weighed and buffer added prior to trituration. Inoculation Procedures All inoculations were performed within 2.5 hours after inoculum preparation, and most were performed on primary leaves using the half leaf method (25). A summary of this method is as follows: Each plant is a block, each half leaf of Opposite primary leaves is a unit, and four treatments in each block of 4 units. Randomized Blocks: Four Treatments in Blocks of Four Units: Plant Number Leaf number 1 2 3 4 5 6 19 Leaf surfaces were dusted prior to rub inoculation with 500 mesh silicon carbide using a hand-operated atomizer. The amount of inoculum was carefully measured with a glass pipette. Generally 2 drops (0.1 ml) of inoculum was applied per half leaf. The drops were placed near the petiole base and distributed over the leaf surface with a pestle; strokes were made to the leaf tip and back again two times to insure uniform distribution of inoculum. After inoculation the rubbed leaves were rinsed with a gentle stream of distilled water from a squirt bottle. Each experiment was repeated at least 3 times, and in each experiment virus purity was determined by inocu- lating 3 plants each of the differential varieties Rainy River (type), Pinto UI-lll (N.Y.-15), and Great Northern UI-123 (V123). When inoculated plants were maintained in the greenhouse, inoculations were made in the afternoon between 2-4 p.m. in order to have comparable conditions. It has been reported that number of local lesions depends on the time of the day that inoculation is done (2, 26). Data Collection In those plants where local lesions were formed, at least two counts were made several days apart. If little or no change in lesion number was noted, no addi- tional counts were made. 20 Experiments Preliminary Attem ts to Reproduce Lesions Reported pngaumeyer and Goth Two pots each of Pinto UI-lll, Plentiful, Potomac, Dade, Monroe, Sanilac, Great Northern UI-31, Great Northern U-123, and Stringless Green Refugee beans were inoculated with BCMV type, V15 and V123 strains at 9 days after planting. Inoculated plants were incubated in growth chambers at 3 different air temperatures (20-24-28 C) and 12 hour photOperiOd. Inoculum was prepared by grinding young leaves with good mosaic symptoms in a mortar with buffer phOSphate pH 7.4 in a ratio of 1:1 (weight of leaf in gm/ml buffer). The inoculation was done by dipping the pestle in the virus preparation and rubbing carborundum- dusted primary leaves. The same experiment was repeated at 24 and 28 C under continuous light and at greenhouse conditions (winter season). Best Method of Inoculation Six Monroe plants, ll-days-old, were inoculated with juice inoculum (1:4) of BCMV1 using the same half leaf method previously described. In one randomized block, the inoculation treatments consisted of: (1) half leaves rubbed with a square of polyurethane foam 3 x 3 cm and 0.5 thick, (2) cheesecloth pad, (3) pestle, and (4) finger. 21 In another randomized block, the treatments consisted of (1) pestle, (2) "Q"-tip, (3) glass spatula, and (4) pestle using "healthy" juice. Inoculumng Phosphate buffers (0.01 m) of different pH values were prepared by altering the ratios of 0.01 M Na HPO 2 and 0.01 m KH2PO4 until the desired pH was obtained. 4 Inocula were then prepared by preparing dilutions (1:4) in the different buffers. Groups of 12 Monroe plants, l2-days-old, were inoculated by the half leaf method (randomized blocks) with BCMV type using the following treatments: A = pH 6.4, B = pH 7.0, C = pH 7.4 and D = pH 7.8. An additional group of 12 Monroe plants was inocu- lated by the same method using the following pH: A1 = pH 5.0, B1 = pH 6.0, C1 = pH 7.4, and D1 = pH 8.5. Relation Between Dilution of Inoculum and Lesion Formation A group of 6 Monroe plants (lZ-days-old) was inoculated with the type strain and another group inocu- lated with the N.Y.-15 strain, using the previously de- scribed pestle method of leaf inoculation. The following dilutions of inoculum were prepared in 0.01 M PO4 buffer of pH 7.4: 1:1, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256. Six half leaves were inoculated with each dilution. 22 In another experiment, the dilution end point of BCMV and V were determined. Inoculum dilutions of l 15 1:100, 1:500, 1:1,000, 1:10,000 were prepared in distilled water and applied to primary leaves as described previously. Relationship Between Age of Leaf and Legion Formation Formation.--Primary leaves on each Of 6 Monroe plants at 8, 9, 10, 11, 12, 13, 14, 15 and 16 days after planting were inoculated with the type strain of BCMV using the half leaf method. Each plant was inoculated with 4 different inoculum dilutions (1:1, 1:4, 1:8, and 1:16). Pre- and Post-Inoculation Light Treatment Because preincubation in darkness, or shading has been shown to markedly affect lesion formation in certain host-virus combinations (12), the following experiments were conducted: 1. In the first experiment the following light- dark regimes were used: (a) Plants growing in normal greenhouse conditions were inoculated and returned to the same greenhouse condition, (b) plants growing in normal greenhouse conditions were inoculated, then incubated 24 hours in dark. (c) Plants preincubated for 24 hours in the dark were inoculated, and then placed in the dark for 23 an additional 24 hours. (d) Plants preincubated for 24 hours in the dark were inoculated, and then placed at normal greenhouse conditions. For each treatment 6 Monroe plants (lZ-days-old) were used, and the concentration of inoculum was 1:4. Darkness during daylight hours was obtained by placing the plants beneath a box (3.5 x 2.0 x 1.5 feet) covered with black plastic. 2. In the second experiment 3 different lengths of preinoculation shading were compared, (a) 48 hours, (b) 24 hours, and (c) 18 hours. After inoculation, all plants were returned to normal greenhouse conditions. Data taken at 7 and 10 days after inoculation included number of local lesions (at 7 days) and lesion diameter (at 10 days- diameter of 10 local lesions per leaf). The Effect of Temperature Primary leaves of three Monroe and three Topcrop bean plants, lZ—days-old, were inoculated with BCMVl, and then held in different growth chambers at constant air temperatures of 16, 20, 24 and 28 C. All chambers were programmed with 14 hr. of light daily, with a light in- tensity of 1,150 ft-c at leaf surface. To compare the local lesions formed by BCMV on Monroe with the hypersensitive lesions formed on Topcrop, 3 plants of each variety were inoculated with the type 24 strain, and subsequently maintained in darkness at 32 C. In some cases detached, young primary leaves (lo-days-old) and trifoliate leaves were also inoculated and maintained under the same conditions in petri dishes containing a small amount of water. CHAPTER I I I RESULTS Preliminary Attempts to Reproduce Lesions Reported By Zaumeyer and Goth Three different kinds of lesions_were obtained in the preliminary experiments. The first kind of lesion (Figure ZB) appeared approximately 7 days after inocula- tion as barely—visible chlorotic spots of diameter about 2 mm. These lesions gradually became necrotic after 2-3 weeks and are referred to as "Ghost Spots" (GS). GS lesions appeared on primary leaves Of Great Northern UI- 123, Great Northern UI-3l, Pinto UI-lll, and Sanilac in— oculated with BCMV and BCMV l 15 temperature regimes (Tables 3, 4). This type of lesion, at different light and however, was not observed consistently, and was not ob- tained in the greenhouse (Table 5) (Figure 3). The second kind of lesion was a necrotic ring spot 1-2 mm in diameter that was clearly visible 5 days after inoculation. These ringspot (RS) lesions (Figure 3B) were observed on primary leaves of Potomac, Rainy River, Plenti— ful, Stringless Green Refugee, and Monroe bean inoculated with all three strains of BCMV (Tables 3, 4, 5). Although 25 26 Figure 2A. Vein necrosis (VN) on Great Northern UI-31 inoculated with BCMVlS. Figure ZB. Ghost Spots (GS) on primary leaves of Great Northern UI-123 inoculated with BCMVl (arrow). 27 Omfiuom MHDcmzvmumcH mfimouomc GHO> n z> Q Homm mcwu H mm mcoflmma Hmooa u an uomm “mosm H mm mfioumfihm on u I . . I . . I . omEo O z> Qmm z> Qmm z> Qmm z> hmm nmm z> nmm Qmm u m I I I I I hmm I hmm I admwucmam I I I I I I z>.nmo I nmw HHHIHD Opcwm z>. mm z>. mm I z>. mm z>. mm I z>. mm z>. mm mm mwmsmmm Q Q Q Q n Q Q comma mmmamcfinum I I I I I I nmw nmw I OOHHsmm z>.mm z>.mm qq z>.mm mm an z>.mm mm on moncoz I I I z>. m0 m3 I z>. m0 z>. mo mo mmHIHD a Q n n n cumcpuoz DOOHO I I I z>. mu z>. m0 I z>. mm z>. m0 m0 HMIHD n a h a Q cumcuuoz pmmuo I I I I I I I I I mono 00mm Oovm Ooom 00mm Oovm Ooom 00mm Oowm Ooom mumflnm> comm MNH> mH> H> damnum >20m .A>20mv msnfl> memoa OOEEOO smog mo mcflmuum m sufl3 Omumasoocfl mw>mma Oman hnmfiflum mo Omcommmu Eoumaxm so AOOHHOO Oponm moon O>Hm3uv muzumummfimu Ham unnumcoo mo pommmm one .m magma 28 OOEHOM haucmswmumcw9 mEOumshm oc mwmouomc me> u 2> 90mm mcwh H mm 902m umo9m n ma M I I 92> 92> mm.92> mm.92> mmmsmmm :OOHO mmmamcflu9m I I I I om Ham 92> 92> H. m . . . OOEO o 92> 92> 92> mm 92> mm 92> mm 92> u m mm.z> mm.z> mm.z> mm.z> mm.2m> mm.z> mouse: I I . I . . I cum Ho OOH 9mO 92> 9mm 92> 92> 9mm Hm HD 99 2 p O I I I I m m 92> 92> O Q Oomm Oowm Oomm O.am comm oovm mumflum> comm MNH> mH> H> cannum >209 m.A>20mv mOHH> OHOmOE GOEEOO :mm9 mo mcwmnum m 9uw3 Umumadoocw mo>mma cmm9 mumefium mo Omcommmn Eoumfimm co Au9mfla msoscwucoov mnzumnmmfimu Ham ucmumcoo mo pommmm O99 .v OH9OB 29 Table 5. Symptom response of primary bean leaves inocu- lated with 3 strains of bean common mosaic virus (BCMV) under greenhouse conditions (Winter season).a BCMV Strain Bean Variety V1 V15 V123 Dade - _ _ Great Northern UI-3l - - _ Great Northern UI-123 — _ _ Monroe LL LL LL Rainy River — Rsb Rsb Sanilac - VNb VNb Stringless Green Refugee RSb - - Pinto UI-lll - _ _ Plentiful — _ - Potomac — _ _ TOpcrOp — - _ ano symptoms LL = local lesions RS = ring spot VN = vei necrosis infrequently formed 30 "- l,"* . A, -— ,_-_.__H_. -177 ,_--_..__C Figure 3A. Local necrotic lesion on primary leaf of Monroe bean. Figure 3B. Ringspot lesion on primary leaf of Monroe bean. 31 infrequently formed on the first 4 of these varieties, RS lesions were always observed on the Monroe variety when the inoculated plants were held at 24-28 C under 12 hr. light or under continuous light. At 28 C, however, the lesions were always accompanied by strong vein necrosis, making it difficult to count them (Figure 2A). The third kind of lesion appeared only on leaves of Monroe bean at 16-20 C (12 hr. photOperiod or contin— uous light) and at greenhouse conditions (winter); these lesions are called local lesions (LL) (Figure 3A). The local lesions were circular and necrotic, well-defined and clearly visible 4-5 days after inoculation. These lesions enlarged in diameter from 0.5 mm at 6 days to nearly 0.8 mm at 10 days after inoculation. Throughout the duration of this study, LL were consistently formed on inoculated primary leaves of Monroe bean maintained either in the greenhouse or in a 20 C growth chamber. Numerous individual local lesions triturated separately and inoculated to susceptible bean varieties induced typical BCMV symptoms. Best Method of Inoculation Of numerous inoculation techniques examined, the pestle appears to give the most consistent and reproducible results (Tables 6, 7). At least a portion of the inoculum applied to each half leaf (0.1 ml) was absorbed when 32 cheesecloth, foam rubber pad, and "Q"-tip devices were used; this absorption could account for the lower lesion counts Obtained with these devices. None of the devices tested caused any appreciable leaf damage. Table 6. The effect of inoculation method on local lesion formation by BCMV1 on primary leaves of Monroe bean. Average Number Number Lesions Lesions Inoculation Method per 6 half leaves per half leaf Cheesecloth pad 83 13.8 i 3.0 Finger 38 6.3 i 2.5 Foam rubber pad 79 13.1 i 2.3 Pestle 182 30.3 i 6.6 Table 7. The effect of inoculation method on local lesion formation by BCMV on primary leaves of Monroe bean. 1 Average Number Number Lesions Lesions Inoculation Method per 6 half leaves per half leaf Frosted glass spatula 102 17.0 i 2.6 Pestle 195 32.5 i 5.0 Pestle-- no inoculum 0 0 H N O G) "Q"-tip 40 6.6 33 Although the data presented in Tables 6 and 7 are from two separate experiments, it is worth noting the con- sistency of lesion counts (30.3 versus 32.5) obtained with the pestle method of inoculation. Also note that the pestle alone (inoculum replaced with water) caused no damage which could be mistaken for local lesions (Table 7). InoculumppH Within the general pH range of 6.4 to 7.8, local lesion counts were quite uniform and comparable (Tables 8, 9). Inocula prepared in buffer of pH 5.0, 6.0, and 8.5 produced fewer local lesions per half leaf than inocula of the above pH levels. Throughout the remainder of this study, all inocula were prepared in pH 7.4 buffer. Table 8. The effect of inoculum pH on local lesion for- mation by BCMVl on primary leaves of Monroe bean. Average Number Number Lesions Lesions Inoculum pH per 12 half leaves per half leaf 6.4 380 31.6 i 4.7 7.0 373 31.0 i 5.1 7.4 371 31.0 i 4.2 7.8 395 32.0 H- h 0 KO 34 Table 9. The effect of inoculum pH on local lesion for- mation by BCMVl on primary leaves of Monroe bean. Average Number Number Lesions Lesions Inoculum pH per 12 half leaves per half leaf 5.0 238 19.5 i 5.8 6.0 304 25.3 i 5.1 7.4 352 29.3 i 4.6 8.5 257 21.4 i 4.5 Relationship Between Dilution of Inoculum and Lesion Formation Local lesion production on inoculated leaves of Monroe is inversely related to dilution of inoculum; i.e., the lower the inoculum dilution, the higher the lesion counts (Tables 10, ll, 12, Figures 4, 5). Note that with both the V1 and V15 strains of BCMV, a straight line rela- tionship exists between local lesion number and inoculum dilution at dilutions between 1:4 and 1:32. The dilution end point of BCMV, as assayed on Monroe by the V1 and V15 strains, lies between 1:1000 and l:10,000 (Table 11). Unless indicated otherwise, inocula were diluted 1:4 in pH 7.4 buffer throughout the remainder of this study. 35 Table 10. The effect of inoculum dilution on local lesion formation by BCMVl and BCMV15 on primary leaves of Monroe bean. Number Lesions per 6 half leaves at several inoculum dilutions BCMV Strain 1:1 1:4 1:8 1:16 1:32 1:64 1:128 1:256 V1 428 205 171 149 121 39 21 18 V15 385 196 159 133 113 43 36 23 Table 11. The effect of inoculum dilution on local lesion formation by BCMVl and BCMV15 on primary leaves of Monroe bean. Number Local Lesions per 6 half leaves at several inoculum dilutions BCMV Strain 1:100 1:500 1:1000 1:10,000 V1 28 15 5 0 V15 52 21 13 0 Relationship Between Age of Leaf and Lesion Formation Age of leaf is an important factor in the production of local lesions by BCMV on Monroe bean (Table 12, Figures 6, 7). With the exception of the data for lO-day-old plants, rather uniform local lesion counts were obtained on plants inoculated 8, 9, ll, 12, and 13 days after planting 36 MOOH MHO9 HOm OGOHOOH HO9EO: OmOHO>O mO>OOH MHO9 m MOO chHmOH HO9E§G O>¢ OZ.m H.m mm H.HH hm m.oa vm m.mN on m.h mv m.m om H.hH MOH H.mN HmH m.mN th m.Hm mom m.hN moa m.mN mmH mana o.mH mm m.mH mm m.mN 55H m.Hm oma m.mm NHN H.mm mmm m.mN NhH H.om Hma mud H.mN bmH m.mH NHH m.mm MAN m.mm mHN m.mm mmm 0.0m owm m.hm mNN m.mN mud vua H m.vm NON m.Nm mma m.ov MON H.mb Hmv m.mHH wan m.hm 5mm H.mh Hmv m.mm NON and 0>AN OZ Obkm OZ Okrdm OZ O>¢ OZ Okr/N OZ 0>AN OZ 0>¢ OZ 0>¢ OZ Our.“ 02 SOH¢5H HO OH ma wH ma NH HH OH m m EDHSOOGH O.mOmO ucOHOmmaO mo OOOOHQ cOO9 OOHOOS mo mO>OOH NHOEHHQ co H>zom m9 wcoamOH HOOOH mo cOfluOEuOm co cOHusHHO mo DOOMMO O99 .NH OH9OB 37 O O P :00.— u0¢20<~ .0 uo>l£ 2505....- ..0 >30. .0 .59.: 03. >9 c3331.... .21... :30. so 53.390... .0 5:2...1 to .9030 O...— co:a:o $3.3»...- Ofl—«p . ‘0"— .v sin-0.. «03 0.3 Cup '3 a; m a. // 2+ A. On I on. I on“ m—>.T UDI can —>run I on. “In; 9 10¢ suolsol |I30| h on season 38 Figure 5. Primary leaves of Monroe bean inoculated with different dilutions of BCMVl. Dilution end point of BCMV is between 1:800 and 1:1000. 139 .cuon mouse: uo oo>uo~ no >xun >9 uous-u occuoon uouoH no non-s: one so cougadav Co “comma use Deanna 40 Figure 6. Seedlings Of Monroe bean at various times after planting. From right, 8, 9, 10, ll, 12 and 13 days after planting. 41 7. ;’14” off ’l"" Wo«¢-¢44... (.1 MW"¢-Ov4\: £¢_* ( W, “’ ‘14 J I. 42 I. 0000 2.0-.02.? .0 3:0..— cooa mom—202 .0 .023. :05...— 00 —>Iun >9 2.0.00. .000. .0 :0_.0E..0. :0 0020...... 50.000... .0 .0030 0.: . a .050‘. 00.00.0000. .0 0.0 000.0 N mfl—aG—m 0. a. I 0. 2 . a. 3 .2. . 0 . _..... ..... II I I I .. ..r.. I/ \flllr 65...”? . I/ \ AI: ............ ... 0: Cl '3' 1 ...Av. ... 30"! /rIIII fz/ [III/1...... ha: / / .. x . . J4 IA... ......0. - 0: // //..... \sss \. / ll ss 0 a / .\ can /I / .\ n' s s / \ . \L 0: /o R‘ .Du— 8... ............... / s\ O l A A 0”” ll /. N _ . 0n0 ....l.l.l . < Oak 100 000.00. 00A00| "III , 43 with virus inoculum diluted 1:4. It is quite difficult to count more than 40-60 lesions per half leaf; consequently, the very high counts recorded for lO-day-old leaves may be subject to error. For this reason, and because many Of the previous studies employed lZ-day—old plants, it was decided to standardize age of bean at inoculation to 12 days after seeding. Pre- and Post-Inoculation Light Treatment Interruption of the normal light conditions which exist in the greenhouse (winter) exerts a great influence on the quality, as well as the quantity of local lesions produced on inoculated leaves of Monroe bean (Tables 13, 14, Figures 8, 9). In terms of number of local lesions, post-inoculation shading (24 hr.) of plants had no effect, while preinoculation shading (24 hr.) increased the lesion counts about 40%. A combination of pre- and post-inocula- tion shading (24 hr. each) reduced lesion counts by about 30%. The Optimum period of pre-inoculation shading was found to be 24 hr.; 48 hr. shading drastically reduced lesion formation. Lesions on leaves shaded prior to inoculation were not only more numerous, but also larger than lesions on leaves under normal conditions (Figures 8, 9). Lesions on leaves receiving the 24 hr. dark-greenhouse treatment were of the ringspot type (RS), averaging 1.56 mm (average of 44 Figure 8. The effect of different light treatments on local lesion formation by BCMV on primary leaves of Monroe bean. (A) Greenhouse-Green- house, (B) Greenhouse-24 hr. dark, (C) 24 hr. dark-24 hr. dark, and (D) 24 hr. dark-Green- house. 45 .0009 uOIZO! .0 00>00. >000...:. 00 {Sun >9 1000.00... 000.00. .000. .0 00.0 00 0.000.000... ...... 00000:... .0 .0030 0...— G ~830:I ...-.05 00.00. .0 00000.0.n n.NIl—.« n.—| —.— O...II0.O 3'80 ...LIIIIII‘IIIIII op I ‘ i i i . l l I 0 fl' I --II. 00 00000000001803. .... t« I W l I at... ... 001...... ... 00 Ema—u I I . 0.00.0 .... ¢NI0000000000D . _. I . I I I I I I, .. III. 00— I-0000..00000I0000..00000 m II 100 000100| 00A00| 46 120 lesions) in diameter; lesions from normal conditions (greenhouse-greenhouse) were of the necrotic local lesion type, averaging 0.88 mm in diameter. Lesion types in the remaining two treatments were ringspot (24 hr. dark-24 hr. dark and greenhouse-24 hr. dark). A combination of ring- spot and local necrotic lesion was found in some leaves with the treatment greenhouse-24 hr. dark. Table 13. The effect of different light treatments on local lesion formation by BCMVl on primary .leaves of Monroe bean.a Number Lesions Average Number Light Treatments Before per 24 Lesions and After Inoculation half leaves per half leaf Greenhouse-Greenhouse 702 29.2 i 4.2 Greenhouse-24 hr. dark 762 31.7 i 3.2 24 hr. dark-24 hr. dark 510 21.2 i 5.1 24 hr. dark-greenhouse 891 37.1 i 3.6 aSee text for greenhouse conditions--all plants inoculated 2-4 p.m. Effect of Temperature on Lesion Formation Air temperature also affects both the quality and quantity of local lesions formed by BCMV on primary leaves of Monroe bean (Table 15, Figures 10, ll, 12). Lesions formed at 16 C and 20 C were completely necrotic, whereas 47 Figure 10. Ringspot-type lesion on primary leaf of Monroe bean inoculated with BCMVl at 24°C. 48 Figure 11. Severe vein necrosis on primary-leaf of Monroe bean inoculated with BCMV1 at 28°C. Local lesions are indistinct and difficult to count. 49 Figure 12. Hypersensitive-type reaction of primary leaf of TopcroP bean inoculated with BCMV1 at 32°C. 50 those formed at 24 C and 28 C were primarily ringspot- shaped. Lesion diameters at these temperatures were 0.2 mm, 0.5 mm, 1.2 mm, and 2.0 mm, respectively. Lesions were clearly visible 5 days after inoculation at 20 C and 24 C; lesions were visible 7 days after inoculation at 16 C. Lesion numbers were uniform at 20-, decreased slightly at 24 C, and decreased markedly at 16 and 28 C. Table 14. The effect of different lengths of pre-inocu- lation shading on local lesion formation by BCMV1 on primary leaves of Monroe bean. Average Number Length of Number Lesions Lesions Shading Period per 24 half leaves per half leaf 48 hr. 230 9.4 i 3.3 24 hr. 895 37.2 i 5.1 16 hr. 843 35.2 i 4.2 0 hr. 689 28.5 i 3.6 The typical hypersensitive reaction of TOpcrop bean was obtained when primary leaves of this and of the Monroe varieties were inoculated with BCMVl (Figure 13) and incubated at 32 C in the dark; hypersensitivity, evi- denced by the formation of necrotic lesions, occurred on both detached and on attached leaves. Topcrop, however, did not form lesions of any type when inoculated with the three strains of BCMV under greenhouse temperatures. {. Figure 13. 51 Appearance of BCMV-induced lesions on inoculated primary leaves of Monroe bean at different times of the year. (A) Fall (October) lesions are Ringspot with necrotic center, (B) Winter (Jan- uary) lesions are local necrotic lesions, and (C) Spring (April) lesions are Ringspot with non-necrotic center. All lesions were formed in the greenhouse. 52 Table 15. The effect of constant air temperature on local lesion formation by BCMVl on primary leaves of Monroe bean. Number Average Lesions Number per 24 Lesions Average Temperature half per Lesion Lesion (0 C) leaves half leaf Appearance Diameter 16 76 3.1 necrotic spot 0.2 mm 20 569 23.5 necrotic spot 0.5 mm 24 339 14.1 ring spot 1.2 mm 28 180a 7.5 ring spot 2.0 mm aLesions indistinct and difficult to count. Thermal Inactivation of BCMV Lesion production by BCMV1 on primary leaves of Monroe bean ceased to occur when the inoculum was heated at 58 C, but not when the inoculum had been heated at 54 and 56 C for 10 minutes. Local Lesion Formation by BCMV_on Trifoliolate Leaves of Monroe Bean BCMV induces local lesion production on inoculated trifoliolate leaves as well as on inoculated primary leaves (Table 16, Figure 14) of Monroe bean. Although total number of local lesions per trifoliolate leaf in- creased slightly with age of leaf, there was very little 53 Table 16. Local lesion production by BCMVl on trifolio- late leaves of Monroe bean inoculated 25 days after planting. Trifoliolate Leafa l 2 3 Leaflet Leaflet Leaflet Plant Position Position Position No a b c a b c a b c l 50 58 41 47 6O 52 49 41 56 2 26 47 37 41 40 39 l3 16 19 3 l3 6 12 10 7 13 3 5 2 4 21 27 22 18 18 25 6 9 8 5 13 15 ll 23 22 17 6 8 5 6 17 15 17 15 16 16 7 9 8 140 168 140 154 163 162 84 88 98 a1 = bottom (oldest) 2 = middle b3 = tOp (youngest) a-c = counterclockwise, looking down 54 Figure 14. Local lesion production on trifoliolate leaves of Monroe bean inoculated with BCMVl. 55 56 variation in lesion counts between the leaflets of the same leaf. There was also very little variation between plants of the same age. Reaction of Monroe Bean to Other Plant Viruses Two additional virus diseases of bean, namely southern bean mosaic (SBMV) and yellow bean mosaic (YBMV) were inoculated onto primary leaves of 9-day-old Monroe bean seedlings, using the previously described method of inoculation. These virus diseases are also commonly found in beans, and therefore represent the viruses most likely to be confused with BCMV. Within two weeks after inoculation, marked symptoms of mosaic were observed on the SBMV-inoculated plants of Monroe bean. In no case were lesions of any type observed on the inoculated primary leaves. Repeated attempts to obtain-YBMV infection in Monroe were negative, as measured both by visual symptoms as well as subinoculations from the Monroe leaves to sus- ceptible bean varieties. Monroe therefore appears resis- tant to the strain of YBMV tested here. CHAPTER IV DISCUSSION Previous efforts by numerous investigators to find an efficient, useful local lesion host for bean common mosaic virus (BCMV) have been only partially successful. Zaumeyer and Goth (49) reported two types of new local lesions in the mid-sixties; one of the lesions, however, was not consistently produced, and consequently was not studied further. These two authors did report the con- sistent production of necrotic local lesions by three strains of BCMV on the leaves of an "unnamed bean cross from Florida." R. A. Conover of Florida who was responsible for making this bean cross was contacted to obtain seed; un- fortunately, Conover neither had seed nor knew anyone from whom seed could be obtained. Instead, he sent seed of Dade bean variety which he considered to be a sister line of the cross used by Zaumeyer and Goth, and which he believed might react in the same way as did the cross. All efforts to obtain lesions on the bean variety Dade with BCMV were fruitless. No combination of tempera- ture and light regime as mentioned by Zaumeyer and Goth 57 58 was successful in stimulating the production of these lesions on Dade. Since seed of the unnamed bean cross is apparently no longer available, and since its closest genetic relative, Dade, did not form local lesions, at— tention was then devoted to other reported lesions and lesion hosts. Although produced only inconsistently, it was thought that a study should be made of the other lesion type reported by Zaumeyer and Goth. Lesions similar to their ghost-like chlorotic spots were formed in the present study (GS lesions) on primary leaves of Great Northerns UI-31 and 123, Pinto UI-lll, and Sanilac inoculated with BCMVl and BCMVlS; these are the same varieties used by Zaumeyer and Goth. The present study confirmed the in- consistent occurrence of this type of lesion on these varieties. Similarly, lesions (RS lesions) were obtained on primary leaves of Potomac, Plentiful, and Stringless Green Refugee inoculated with all three BCMV strains in the present study. Such lesions are considered similar to the round, white necrotic ringed lesions described by Zaumeyer and Goth (48). No combination of temperature and light regime overcame the inconsistency of formation of these types of lesions. Another local lesion host for BCMV is thought to be the bean variety Monroe, a Navy (pea) type bean released by Cornell University some years ago. Although apparently 59 resistant to the V1 and V15 strains of BCMV, Monroe devel- Oped local lesions when inoculated with the V strain 15 (Dr. A. L. Andersen, unpublished information). Andersen noted that single plants of Monroe did not react identic- ally, and he therefore made single plant selections for local lesion reaction. In the present study we used the bulked seed of Andersen's single plant selections as well as commercial Monroe seed. During the course of this study, consistent pro- duction of well-defined lesions have been obtained on primary leaves of Monroe grown at temperatures below 28 C and at different light conditions (continuous and 12 hr. photOperiod). Such lesions were easily counted visually, and were different from the "hypersensitive" lesions which develOp on Topcrop and other varieties which possess dominant resistance to BCMV derived from Corbett Refugee (40); hypersensitive lesions are formed only at 32 C. It therefore appears that Monroe responds to BCMV inoculation on the primary leaves, as well as the trifoliolate leaves, by the production of local lesions of a type not previously reported. That the lesions formed on inoculated primary leaves of Monroe are due to BCMV is supported by several lines of evidence. Firstly, juice obtained from healthy plants does not induce lesion formation when rub-inocu- lated to primary leaves of Monroe. Second, single local 60 lesions removed from inoculated Monroe leaves induce typ- ical BCMV systemic symptoms on susceptible bean varieties. Third, lesion production on inoculated primary leaves occurs if inoculum is heated at 56 C for 10 minutes, but does not occur if inoculum is heated at 58 C for 10 min- utes; the thermal inactivation point for BCMV is reported to be between 56 and 58 C (38). Fourth, 5 different iso- 2 of V lates of BCMV, 2 of V and l of V123, all in- l' 15' duced these lesions when inoculated to Monroe. Recognizing the potential importance of Monroe as a new local lesion host for bean common mosaic virus, the author has studied several of the more commonly-recognized factors which are known to affect local lesion assays. The most preliminary of these factors is perhaps the method of inoculation to be used in all subsequent work. Although most workers have used pads of cheesecloth or some other absorbent material such as cotton or foam rub- ber, the rough edge of the pestle seemed to give the most uniform and efficient results in this study. One of the advantages of this method of inoculation is that a half leaf can be inoculated uniformly with a rather small amount of inoculum. This uniformity can be evidenced by the consistent production of 28-35 lesions when 0.1 ml (2 drOps) of juice inoculum (1:4 dilution) was applied to half leaves. Another advantage is that the pestle is routinely sterilized with the mortar for the preparation 61 of inoculum, and is therefore immediately ready for use as an inoculator after triturating the tissue for inoculum. Another equally important factor preliminary to studying the local lesion assay of a plant virus is the inoculum itself. Many plant viruses are particularly sus- ceptible to inactivation because of adverse pH conditions (26). BCMV appears to be quite stable as assayed by local lesion formation when prepared in 0.01 M NazHPO4-KH2PO4 solutions buffered at pH 6.4-7.8. Virus inactivation, as measured by fewer local lesions on Monroe leaves, begins to occur at pH 6.0 and 8.5. BCMV therefore appears to be quite stable to a rather wide range of pH. To be useful as a tool for quantitative measurement of a plant virus, there should exist a direct relationship between number of local lesions and amount of virus in the inoculum. This information is usually obtained by plotting lesion number versus dilution of inoculum, and ascertaining that portion of the graph which represents a rather straight line (25), i.e. where infectivity as measured by number of lesions is directly proportional to dilution. BCMV assayed on Monroe bean leaves produces a characteris- tic S-shaped curve when number of lesions is plotted against dilution of inoculum (Figure 4); a straight line relationship exists between dilutions of 1:4 and 1:32. At dilutions higher than this, lesion numbers change very rapidly. At dilutions of 1:4 and 1:8, lesion counts per 62 half leaf are in the range of 20-35 where there is little possibility of making a counting error. With the exception of lO-day-old plants, Monroe responds with a uniform number of local lesions when in- oculated with BCMV at 8-12 days after planting. In at least three separate experiments, lesion production was greatest at 10 days after seeding, decreasing at 8, 9, 11, and 12 days. Because plants grow at different rates when maintained at different temperature conditions, it was felt that the period 9-12 days would be most useful to other investigators who might be studying the BCMV-induced lesions on Monroe. For example, investigator "A" may grow his plants at 24 C and inoculate at 10 days, while in- vestigator "B" grows his plants at 28 C and also inocu- late at 10 days. Light conditions before and after inoculation are known to exert a strong influence on the number and the type of lesions formed in certain host-virus combinations (12). In the present study preinoculation darkness for 24 hr. increased lesion production by BCMV on leaves of Monroe. Such stimulation of lesion production has also been reported for tobacco necrosis virus (TNV) and for alfalfa mosaic virus (potato calico strain) on bean leaves (11). This similarity of response by three different local lesion-inducing viruses would suggest that pre- inoculation shading results in a general increase in 63 susceptibility of bean leaves to lesion formation. It would be interesting to examine the reaction of Monroe to TNV and alfalfa mosaic virus. Preinoculation shading of plants in the dark for 48 hr., however, had a deleterious effect on lesion production by BCMV on Monroe; such is also the case with TNV on bean (5). This would suggest that, among other factors, the photosynthetic economy of the host at the time of inoculation is important in the formation of lesions (41); 48 hr. of shading resulted in leaves showing moderate chlorosis, whereas 24 hr. of shad- ing did not. Lesion size is another character which is affected by the condition of light before and after inoculation. In the present study, larger lesions were consistently obtained when leaves were shaded for a 24 hr. period prior to inoculation with BCMV. Best (6) using Nicotiana tabacum-TMV and Helms (20) using bean-TMV and Desjardins (12) working with alfalfa mosaic virus on bean, all report that size of lesion produced on leaves was greatest when the leaves were preincubated in the dark prior to inocula- tion. The unusual feature of the larger lesion in the BCMV situation is that the lesion quality was altered; that is to say, the lesions assumed the shape of a ring- spot, rather than the shape of a totally necrotic lesion. The reasons for this alteration in lesion appearance by different light conditions are unknown. 64 Air temperature is another environmental factor which is known to influence local lesion production in plant virus assays (24). Lesion numbers of BCMV on Monroe bean were greatest at 20 C, less at 24 and 16 C, and least at 28 C. These results are similar to those of Crill, gt El. (7) who reported 20-24 C as the optimum temperature range for alfalfa mosaic virus-induced lesions on Bountiful bean. Harrison (19), however, found that TNV lesions on bean plants were most numerous at 10 C, followed by 14, 18, 22, and 26 C. This suggests that the effect of air temperature will vary depending on the virus being as- sayed. The time of lesion appearance after inoculation was affected by temperature also. Lesions were first observed at 20 and 24 C, then 28 C (indistinct), and finally, after several days, at 16 C. Best (6) reported that a drop in temperature from 20 to 15 C delayed the appearance of lesions in the two following plant-virus combinations: Nicotiana glutinosa XE TMV, and N. glutinosa vs tomato spotted wilt virus. Lesion size, as well as number and time of appear- ance, was affected by the temperature at which inoculated plants were maintained. Lesions at 16 C were quite small, normal size at 20, slightly larger at 24 C and 28 C. Desjardins noted the opposite response of bean leaves to AMV; lesions decreased in size as temperature increased from 25 to 30 to 32 C (12). 65 Notwithstanding the various effects which light conditions, air temperature, and age of plant have on lesion formation, the present study suggests that Monroe bean is a superior local lesion host for bean common mosaic virus (BCMV). This bean variety responds to rub- inoculation with several strains of BCMV by the production of local lesions, either ringspot or totally necrotic, which have never been described for this virus on bean. The results of this study suggest that the Monroe bean variety will come to be a very useful tool in the study of BCMV, as well as in the study of virus properties such as replication of virus particles and strain specificity. CHAPTER V SUMMARY A study has been conducted on the formation of local lesions by bean common mosaic virus (BCMV) on primary leaves of various bean varieties. Of numerous varieties tested, only Monroe bean consistently reacted to BCMV inoculation by the formation of local lesions. The lesions formed on Monroe are different from and appear superior to all other previously described lesions induced by BCMV on bean. ¥Of several inoculation devices tested, the rough end of a pestle seemed to give the most uniform and repro- ducible lesion counts on inoculated primary leaves of Monroe bean.‘xInocula of BCMV prepared in buffers of pH 6.4-7.8 were of uniform infectivities as assayed by local lesion counts. Number of local lesions per half leaf was inversely related to dilution of inoculum; Optimum inoculum dilutions of 1:4-1:16 are suggested for routine local lesion assays of BCMV. Preinoculation shading for 24 hr., but not for 48 hr. resulted in better lesion development (number and shape) by BCMV on primary leaves of Monroe bean; such 66 67 lesions were of a ringspot rather than a totally necrotic type. Ringspot-type lesions were also formed on inoculated plants incubated at 24 C and 28 C. Lesion development was comparable on plants inoculated 8, 9, ll, 12, and 13 days after planting; lO-day-old plants were the most sensitive to local lesion formation. This study has found the following conditions to be Optimum for local lesion assay of BCMV on primary leaves of Monroe bean: (1) Plant age-8-l3 days-old, (2) Inoculum-1:4 dilution in pH 6.4-7.8 phosphate buffer (0.01 M), (3) Inoculation device-pestle, (4) Growing con- ditions-20-24 C, 12 hr. photoperiod or natural’light; greenhouse or growth chamber, (4) Preinoculation dark period-24 hr., and (5) Rinsing of inoculated leaves. REFERENCES 10. REFERENCES Andersen, A. L. Observation on bean diseases in Michigan during 1949 and 1950. Plant Disease Reptr. 35:89-90, 1951. Bawden, F. C. "Plant viruses and virus diseases." 4th edition. The Ronald Press Company, New York. 361 pp., 1964. Bawden, F. C. and B. D. Harrison. 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