i H3 
 V 1 
 
 -128 
 
 PODBLIGHT OF THE LIMA BEAN CAUSED BY 
 DIAPORTHE PHASEOLORUM 
 
 by 
 
 L. L. HARTER 
 
 Reprinted from JOURNAL OF AGRICULTURAL RESEARCH 
 
 Vol. XI, No. 10 : : : : Washington, D. C, December 3, 1917 
 
 A THESIS SUBMITTED TO THE GRADUATE 
 FAPULTY CF GEORGE WASHINGTON UNIVERSITY 
 FOR THE DEGREE OF DOCTOR OF PHI UOSOPH* 
 
 PUBLISHED BY AUTHORITY OF THB SECRETARY OF AGRICULTURE, WITH THB COOPERATION 
 OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS 
 
 WASHINGTON : GOVERNMENT PRINTING OFFICE : 1917 
 
.B3H3 
 
 PODBLIGHT OF THE LIMA BEAN CAUSED BY 
 DIAPORTHE PHASEOLORUM 
 
 By L. h. Harter, 
 
 Pathologist, Cotton, Truck, and Forage Crop Disease Investigations, Bureau of Plant 
 Industry, United States Department of Agriculture 
 
 INTRODUCTION 
 
 The Lima bean {Phaseolus lunatus L.), compared with some of the other 
 truck crops, is relatively unimportant from a commercial standpoint. It 
 is grown in a small way in the garden by nearly every farmer, and on a 
 commercial scale in a few sections of the United States, particularly 
 along the Atlantic seaboard and along the Pacific coast. Like many 
 other crops grown only for home use or on a commercial scale in a limited 
 area, its several pests and diseases have either been overlooked or ignored. 
 The Lima bean, however, has its share of diseases, some of which have 
 been fairly well studied, while little is known about others. To the 
 latter class belongs the podblight, a disease of considerable economic 
 importance some seasons in commercial fields. It was first discovered 
 by Halsted (16) 1 in 1891, in New Jersey. Further than this nothing is 
 known as to the time and place of the origin of the disease. 
 
 There is no evidence from the search of the literature that the disease 
 occurs outside of the United States, although it is impossible to state 
 definitely that it does not, in view of the great number of species of 
 Phoma, Phyllosticta, and closely related genera under which the casual 
 fungus may have been described. A careful search through the descrip- 
 tions of species of the genera Phoma, Phyllosticta, and Phomopsis on 
 different species of Phaseolus has revealed none located in foreign coun- 
 tries identical with the organism causing the podblight. It therefore 
 appears safe to assume at the present time that the disease is indigenous 
 to the United States. 
 
 To judge from published accounts, Halsted (16) was the first to recog- 
 nize this disease and found it causing much damage to the pods of pole 
 Lima beans in New Jersey in the fall of 1891. He attributed it to a 
 species of Phyllosticta, pointing out at the same time that large blotches 
 were also produced on the leaves. That he studied the disease with some 
 care and did not content himself with a few passing observations is 
 evident from the fact that he found that the seeds from infected pods, 
 when placed under a suitable environment, such as between moist cloths 
 or paper, developed the fruiting bodies of the fungus. 
 
 1 Reference is made by number (italic) to 'i' Lterature cited," p. 302-504. 
 
 Journal of Agricultural Research, Vol. XI, No. 10 
 
 Washington, D. C. Dec. 3, 1917 
 
 kx Key No. G— 128 
 
 (473) 
 
474 Journal of Agricultural Research vol. xi, No. 10 
 
 In 1892 Ellis and Everhart {15, p. 158) evidently collected the same 
 organism on the pods of Phaseolus lunatus at Newfield, N. J., and de- 
 scribed it as a new species of Phoma, Phoma subcircinata, pointing out 
 that it differed from Phoma leguminum West, in the subcircinate arrange- 
 ment of the pycnidia and the rather larger binucleate sporules. Halsted 
 later apparently concurs with Ellis and Everhart that this is a new 
 species of Phoma, for in 1901 he (27) refers to the disease again by that 
 name and enlarges his earlier description. Two illustrations are ap- 
 pended to his article, one showing the characteristic appearance of the 
 disease on the pods and the other on the leaves, both typical of the trouble 
 as we know it at the present time. 
 
 DISTRIBUTION, PREVALENCE, AND LOSS 
 
 For 14 years after this disease was found in New Jersey it had either 
 not appeared in any other State or had not been discovered. In 1905, 
 however, Clinton (5, p. 265) reports a leafspot of Phaseolus lunatus at 
 New Haven, Conn., apparently the same as that described and illustrated 
 by Halsted. He states that he did not observe the disease on the pods, 
 but adds that it had not been looked for there. Although agreeing 
 macroscopically with Halsted's description of the disease on the leaves, 
 Clinton entertained some doubt of its identity with Phoma subcircinata 
 E. and E. because of the fact that the spores averaged larger (5 to 12 
 by 2.5 to 3.5 n) and were occasionally septate. He suggests the pos- 
 sibility that the fungus may be Ascochyta phaseolorum Sacc. 
 
 In 1 91 2 Cook (6, p. 51J-518) reports a severe outbreak of a disease of 
 pole Lima beans in the State of New Jersey caused by Phyllosticta sp., 
 which occurs on leaves and to some extent on the pods and stems. The 
 following year it was less severe (7, p. 801), but in 1 914 he (8, p. 472) 
 reports the disease again as causing much damage, attributing it to 
 Phoma subcircinata E. and E. A leafspot caused by Phyllosticta phaseo- 
 lina Sacc. was also commonly met with. 
 
 In view of the relatively few times this disease has been reported 
 during a period of 25 years, it is evident that it has generally been either 
 overlooked or disregarded. This is probably due to the fact that the 
 crop is relatively a minor one and that the disease appears usually after 
 the low prices do not justify further pickings. It, however, is much 
 more widely distributed than the published reports indicate. It was 
 reported (unpublished) to the Plant Disease Survey from one locality 
 in North Carolina by R. H. Fulton in 191 3. The writer received and 
 studied specimens of badly infected plants from Maryland in 1914 and 
 1 91 5, and from Virginia in 191 5. During the seasons of 191 4 and 191 5 
 the disease also caused heavy loss to the crop in New Jersey. In con- 
 clusion, it may be stated that the disease is rather widely distributed 
 along the Atlantic seaboard and causes considerable damage to the crop. 
 
 Gift 
 >rgity 
 NOV 20 I8ti 
 
Dec. 3,1917 Podblight of Lima Bean 475 
 
 DESCRIPTION OF PODBLIGHT 
 
 Although the ascogenous stage of this fungus has been connected by the 
 writer with the pycnidial stage, the latter is alone concerned in the injury 
 to the crop in the field. A macroscopic description of this disease there- 
 fore will be confined to the pycnidial or imperfect stage. In the field 
 under suitable conditions the disease first appears on the leaves of plants 
 from 1 to 2 feet high. Wet weather is conducive to the spread and warm 
 weather promotes the development of this as well as many other dis- 
 eases of this type. Serious outbreaks have often occurred immediately 
 following a few days of rainy, warm weather in fields where previously 
 the disease was scarcely noticeable. In such a case the loss is often se- 
 rious; but if dry weather follows new growth of leaves and fruit will be 
 produced comparatively free of the disease. If, however, the weather 
 continues wet, a new outbreak is liable to occur. 
 
 The leaves function as host for the fungus during the earlier part of the 
 season, from which it spreads later to the pods. Large, subcircular, 
 brown, often bordered patches are produced on the leaves (PI. 42, A). 
 These patches, varying in size naturally with age, often attain a diameter 
 of 1 to 3 cm. Infection is not restricted to any portion of the leaflets 
 but the regions bordering the midrib are most frequently attacked. The 
 fungus spreads more or less in all directions from the point of infection, 
 but may often be delimited by a large vein or midrib. In this moribund 
 tissue, or in tissue soon after it is killed, the fruiting bodies (pycnidia) of 
 the fungus break through the epidermis. They are arranged more or 
 less concentrically, and appear first as gray or grayish raised pimples, 
 which later darken and become nearly black. They therefore may be 
 seen as conspicuous, minute black specks on the upper surface of the 
 leaves standing apart, sometimes confluent, on a brown background. 
 The dead tissues finally become dry and fall out, leaving a ragged hole. 
 
 This disease must not be confused with a leafspot of Lima beans and 
 other varieties of beans, as well as of cowpeas, caused by Phyllosticta 
 phaseolina Sacc, the spots of which are smaller, more nearly round, and 
 the pycnidia smaller and fewer in number. 
 
 The disease appears on the pods usually the latter part of July or early 
 in August, at a time when the vines have nearly reached their full devel- 
 opment. Under field conditions it is not certain at what stage in the de- 
 velopment of the pod infection takes place. Young pods are rarely 
 found diseased, and inoculation experiments have shown that they are 
 infected less easily than ones nearly matured. The disease progresses 
 rather slowly, requiring a week or 10 days after inoculation to produce a 
 spot of any size or for the production of pycnidia. The growth of a bean 
 pod is comparatively rapid, and it is likely that infection may take place 
 when the pods are young, the fungus becoming visible as the pod ap- 
 proaches maturity. Infection occurs at any point on the surface of the 
 
476 Journal of Agricultural Research vol. xi, no. «/ 
 
 pod, but more frequently at or near the ventral suture (PI. 42, B). If 
 infection occurs near or at the dorsal or ventral suture, the fungus spreads 
 in all directions in a circular or semicircular manner, the hyphae growing 
 among the cells of the pod below the epidermis. The extent of the hyphal 
 growth of the fungus among the cells is indicated by the darkening of the 
 pod above the invaded area, evidently the first signs of the exhaustion of 
 the food supply and approaching death of that part of the pod. This is 
 soon followed by numerous, minute elevations over the surface, the points 
 where the pycnidia are forming. These lifted points are at first gray, but 
 as soon as the pycnidia break through the epidermis they become nearly 
 black and protrude in a domelike manner from the surface. They are 
 often, though not always, arranged concentrically and stand apart ; or two 
 or more may be confluent in a cluster or chainlike manner. Upon the 
 death of the pod the fungus grows rapidly through the tissues, the 
 pycnidia breaking out more or less over the entire surface (PI. 42, C). 
 The fruiting bodies which are formed subsequent to the death of the pod 
 are usually not formed concentrically. 
 
 Under field conditions the fruiting bodies of the fungus are often found 
 on the dead and more rarely on moribund stems. It is exceedingly 
 uncommon to find typical infections of living stems, and the writer has 
 never been able to produce infection of living stems artificially. The 
 pycnidia, however, appear abundantly after the death of the vine on 
 stems that have been sprayed with spores of the fungus. 
 
 The disease is much more common and destructive on the pole Lima 
 beans than on the bush Limas. 
 
 ETIOLOGY 
 
 The podblight of Phaseolus lunatus is caused by a fungus pathogen to 
 which various names have been given at different times. The use of 
 different names is largely due to the lack of a knowledge of the life his- 
 tory of the fungus and to the practice of many mycologists in separating 
 the genera Phyllosticta and Phoma, respectively, according to whether 
 the fungus occurs on the leaf, or on the stem, or on other parts of the 
 host. 
 
 Halstead (16) first reported this disease in 1891 as causing considerable 
 damage to the pods and leaves, and attributed it to a species of Phyllo- 
 sticta. He was evidently in doubt as to the species, for he does not 
 refer it to Phyllosticta phaseolina Sacc. (30, p. 149), which had been 
 described some years earlier and which occurs commonly on Lima beans, 
 on Phaseolus diver sifolius, Kansas (Kellerman), New Jersey (Ellis), 
 Canada (Dearaess) on Phaseolus perennis, Missouri (Galloway) and on 
 cowpea, Kansas (D. B. Swingle). In 1905 Smith (33) proved the patho- 
 genicity of this organism on the leaves of Lima beans and other varieties, 
 such as kidney and wax, and on cowpeas. He found that the pods 
 were not affected. 
 
Dec. 3. 1917 Podblight of Lima Bean 477 
 
 That the writer has been working with the same fungus which Ellis 
 and Bverhart described as Phoma subcircinata there is little doubt. 
 Material, collected by Dr. J. B. Ellis at Newfield, N. J., and identi- 
 fied by him as Phoma subcircinata, has been examined and found 
 to be identical with material collected at Vineland, N. J., only 4 
 miles distant from Newfield. It should be mentioned in this connec- 
 tion that the material from which Ellis and Everhart made their de- 
 scription was likewise collected at Newfield, in 1892. In 1890, three 
 years before Ellis and Everhart's description of Phoma subcircinata 
 appeared (15, p. 158), Ellis collected and identified as Phyllosticta 
 phaseolina some material from Newfield which upon examination the 
 writer has found to be identical with material later identified by him 
 (Ellis) as Phoma subcircinata and also identical with the material the 
 writer has been studying. 
 
 Phoma subcircinata E. and E. was described by Ellis and Everhart 
 (15, p. 158) as follows: 
 
 On pods of Lima Beans, Newfield, New Jersey, October, 1892. 
 
 Perithecia subcuticular, 70-90 m diam., sublenticular, subconfluent pierced above, 
 membranous, black, subcircinately arranged in large (1 cm.), round, faintly zonate 
 spots, finally spreading and occupying the entire surface of the pods. Sporules 
 oblong-elliptical, hyaline, 2-nucleate, 5-6 by 2-2.5 M. on simple basidia rather larger 
 than the sporules. 
 
 This description fits perfectly the fungus the writer has been studying, 
 with the exception of the size of spores and pycnidia. An examination 
 of the material collected and identified by Ellis as Phoma subcircinata 
 shows the spores to be somewhat larger (6.4 to 8.0 by 2.4 to 3.6 ix, average 
 7.4 by 2.95 fj.), and the pycnidia considerably larger (142 to 276 /*, average 
 185.8 fi). Material collected and identified by Ellis three years earlier as 
 Phyllosticta phaseolina and which he probably later took for Phoma sub- 
 circinata, bore spores (6.0 to 8.0 by 2.8 to 3.2 /*, average 7.2 by 2.88 /*) 
 and pycnidia (197.5 to 260.0 /*, average 219.0 tx) of about the same size. 
 On material which the writer has collected and from which isolations 
 have been made for inoculation work the spores varied from 6.0 to 8.6 
 by 2.4 to 4.1 v, average 7.50 by 3.23 /x, and the pycnidia from 158.0 to 
 475.0 n, average 245.86 fi. From the data at hand it appears evident 
 that the fungus described by Ellis and Everhart as Phoma subcircinata 
 is the same as the one the writer has been studying. 
 
 In view of the fact that the fungus causing podblight has been con- 
 nected with a perfect stage, the genus to which the imperfect form should 
 properly belong is of no great consequence, but it may be of interest to 
 know that this fungus, like a number of others which have been classed 
 as Phoma, belongs to the form genus Phomopsis. In morphological 
 structures the podblight fungus is identical with similar structures of the 
 genus Phomopsis as laid down in Diedicke's (13) revision of the group — 
 
478 Journal of Agricultural Research VoLxi,No. 10 
 
 that is, chambering of the pycnidia, formation of sclerotial stroma, 
 presence of pycnospores and stylospores, and structure of the pycnidial 
 wall. No stroma is formed by this fungus on the leaves. If this fact 
 should be disregarded and Saccardo's classification followed, it should b& 
 classed as a Dothiorella or a Fusicoccum. Stylospores have been found to 
 be abundant in most of the material collected and studied, but none were 
 found in the herbarium material collected and identified by Ellis as 
 Phoma subcircinata. However, isolations have been made from material 
 which did not bear stylospores but developed them in cultures later, 
 which shows that, although not always present, the fungus may pro- 
 duce them. On the other hand, the organism isolated from specimens 
 bearing stylospores do not always produce them in culture. This fun- 
 gus is very similar to a large group of other organisms which have long 
 been classed as Phoma. In a taxonomic study of the group Diedicke 
 (13) transferred a number of species of Phoma to the genus Phomopsis. 
 The podblight fungus does not differ essentially morphologically from 
 the conidial stage of Diaporthe batatatis (E. and H.) Harter and Field, 
 which was first described as Phoma batatae but found later by Harter and 
 Field (21) to be a species of Phomopsis. It is similar also to the organism 
 causing fruitrot, leafspot, and stemblight of eggplant, which was known as 
 Phoma solani on the stem and fruit and as Phyllosticta hortorum on 
 the leaves, both of which were found by Harter (20) to be caused 
 by the same organism and to belong to the form genus Phomopsis. 
 
 Some years before Phoma subcircinata was described by Ellis and Ever- 
 hart, Cooke and Ellis (0, p. 95) described Sphaeria (Diaporthe) phaseo- 
 larum occurring on bean stalks as follows: 
 
 Gregaria, tecta. Peritheciis globosis, immersis, minimis. Ostiolis spinaeformibus, 
 atris, erumpentibus, Ascis clavatis. Sporidiis lanceolatis, quadrinucleatis. Spo- 
 ridia 0.016 mm. 
 
 In the original description no type location was given; neither is it 
 apparent in this or subsequent descriptions whether the fungus was found 
 on Lima beans or other varieties. In the absence of these facts, together 
 with the imperfect description, it would be difficult to identify correctly 
 the fungus if it had not been more fully described later by Ellis and 
 Everhart (14, p. 460) as follows : 
 
 Diaporthe phaseolorum (C. & E.). 
 Sphaeria phaseolorum, C. & E. Grev. VI, p. 93. 
 Diaporthe phaseolorum, Sacc. Syll. 2635, Cke. Syn. 2423. 
 Exsicc. Ell. N. A. F. 188. 
 
 Perithecia gregarious, buried, very small. Ostiola spine-like, slender, projecting 
 for %-yi. mm. Asci clavate, 30-35 by 6-7 /*• Sporidia biseriate, oblong-lanceolate, 
 4-nucleate, scarcely or only slightly constricted, 10-12 by 3 m (16 /x long, Cke.). 
 
 On decaying bean vines left exposed through the winter, Newheld, N. J. Mostly 
 around the nodes of the stem, the surface mostly blackened and the stroma limited 
 within by a black line. 
 
Dec. 3, i 9 i 7 Podblight of Lima Bean 479 
 
 The writer collected a quantity of Lima beans (pods and stems) at 
 Vineland, N. J., in October, 1914, and January, 1915, which bore an 
 abundance of pycnidia of the podblight fungus, generally known as 
 Phoma subcircinata E. and E. After isolating the causal fungus the ma- 
 terial was wintered out at Washington, D. C, and covered with leaves and 
 dirt. On June 2 perithecia of the genus Diaporthe were found to be 
 abundant on stems and pods of this material. The perithecia were 
 scattered and buried in the tissue of the host except the beaks which 
 were long and projecting and somewhat bent as shown by Plate 43, D. 
 A pycnidial fungus was isolated from single ascospores which was iden- 
 tical with the one isolated from the material before it was wintered out 
 Subcultures of this strain were used in inoculation experiments and pro- 
 duced infections identical with that produced by subcultures from iso- 
 lations of single pycnospores. This strain (known as 598), as well as 
 others isolated directly from pycnospores, produced stylospores. The 
 perithecial stage has never developed in culture. 
 
 The perithecia produced on the wintered-out material varied in size 
 from 158.0 to 355.5 fj., average 251.9 n; the asci 28.0 to 46.2 p. by 5.2 to 
 8.0 (x, average 37.4 by 6.73 ju; the ascospores 6.4 to 12.0 n by 2.3 to 4.0 n, 
 average 9.5 by 2.93 /jl. The perithecia of type material varied in size 
 from 158.0 to 237.0 /u, average 215.6 n; the asci 28.0 to 44.0 it by 4.8 to 
 8.0 /x, average 33.6 by 7.0 li; the ascospores 8.0 to 12.0 by 2.4 to 4.0 ju, 
 average 10.0 by 3.3 ju. From the fact that the ascospores, asci, and peri- 
 thecia together with other morphological characters of the two organisms 
 are practically identical, it is concluded that they are the same fungus and 
 should be known as Diaporthe phaseolorum (C. and E.) Sacc. 
 
 MORPHOLOGY 
 
 PYCNIDIAL STAGE 
 
 From the point of infection the hyphae grow in all directions, invading 
 practically all classes of cells except the bast, a layer of which (fig. 1, 
 A, B, d) is found just below the epidermis. Figure 1, A and B, c, shows 
 parenchyma cells traversed by the hyphae. Soon after the death of the 
 cells the pycnidia begin to form, and develop very rapidly thereafter. 
 The hyphal growth increases in the cavities below the stomata or in the 
 intercellular spaces. From here it passes up between the bast cells which 
 are often somewhat separated beneath the stomata. As growth increases, 
 gnarls of hyphae accumulate under the stomata and between the 
 epidermis and the bast cells (fig. 1, A, B, b). From this point the fungus 
 spreads out just beneath the epidermis and develops a more or less 
 circular plate, the base of which rests on the bast cells. Figure 1 , A, shows 
 an early stage in the development of the pycnidial plate. As the fungus 
 growth continues, the epidermis is gradually elevated (fig. 1, B, a) as the 
 
480 
 
 Journal of Agricultural Research 
 
 Vol. XI, No, io 
 
 result of the pressure below. Definite cell layers of the pycnidium are 
 laid down about this time or a little earlier. The pycnidium completes 
 its full development soon thereafter (PI. 43, B). 
 
 The inner layer of the pycnidium is composed of hyalin cells from 
 which the conidiophores arise. A slight cavity is formed at the center 
 concomitant with the release of pressure by the rupture of the epidermis. 
 Inclosed in this cavity numerous conidiophores are developed, extending 
 toward the center from which the spores are cut off. As the spores 
 increase in quantity, the cavity enlarges by the pushing back of the cells 
 lining the cavity and probably by further lifting of the upper part of the 
 pycnidium. It has not been possible to determine definitely when the 
 opening from the pycnidial cavity to the outside through the beak is 
 formed, but it probably takes place only after the pressure within has been 
 greatly increased by the continued production of spores. It is believed, 
 
 Fig. 1. — Diaporthe phaseolorum: A, An early stage in the formation of a pycnidium. The epidermis (a) 
 is not yet disturbed but the hyphae (6) have accumulated directly beneath it. Note the coursing of 
 the hyphae through the parenchyma cells (c) . X130. B, A more advanced stage of figure A. Note 
 the bulging of the epidermis (a). X 130. 
 
 however, that their expulsion is largely brought about through the ab- 
 sorption of water by the gelatinous mass in which the spores are em- 
 bedded, a condition which has been found by Stewart (54) to occur for 
 Guignardia aesculi (Pk.) Stewart. 
 
 The writer does not wish to leave the impression that the pycnidia 
 develop only in the stomatal chambers, but that this is a common and 
 natural place for their formation there can be no doubt. Even in young 
 pycnidia it is not always easy, and sometimes is impossible, to tell 
 whether or not they were being. developed just beneath a stoma. The 
 bast cells of the pods of the Lima bean are very thick walled and are 
 generally pressed so close together that it would be difficult, if not impos- 
 sible, for the fungus to pass between them. These cells, however, are 
 separated at certain places, and often just beneath a stoma, so it would 
 be logical that they should form in such a cavity. From these facts it is 
 evident that, in many cases at least, the pycnidium is developed beneath 
 a stoma, and also at such other places where the bast cells are separated. 
 
Dec. 3, 191 7 
 
 Podblight of Lima Bean 
 
 481 
 
 -G 
 
 Fig. 2. — Diaporthe phas- 
 eolorum: Long, slender, 
 fragile sporophores with 
 two pycnospores at- 
 tached, (a) Sporo- 
 phores; (6) pycno- 
 spores. X500. 
 
 The pycnidia vary greatly in size (158.0 to 475 fi, average 245.86 n 
 from host). They are irregular in shape, chambered with an inner 
 hyalin layer from which the conidiophores arise, and a dark outer layer 
 much thickened and darkened above (PI. 43, B, C), all characters typical 
 of the genus Phomopsis. Sometimes the contiguous sidewalls of two or 
 more pycnidia are ruptured, forming a single cavity (Pi. 43, E). On the 
 other hand, they may be pressed together but separated by a single dark 
 wall and their respective hyalin inner layers, thereby 
 forming a chain of pycnidia (PI. 43, C). The pyc- 
 nidia can hardly be said to be embedded in a true 
 stroma. 
 
 The pycnidia on the leaves and stems are lenticu- 
 lar in shape, rarely, or not at all chambered (PI. 43, 
 A), fewer in number, and smaller than on the pods 
 and usually isolated. They vary in size from 197.5 to 
 260.0 /x, average 219.0 //. 
 
 The pycnospores are borne on slender, hyalin, sim- 
 ple conidiophores (fig. 2, a), i}4 to 3 times the length 
 of the pycnospore. They are mostly oblong to fusoid 
 (fig. 2, b; 3), hyalin, 1 -celled, usually straight, seldom slightly curved, 
 with two large oil droplets. These droplets, sometimes pressed to- 
 gether in the center of the spore, give the appearance of a septum, a 
 characteristic common to the genus Phomopsis and so deceptive that 
 at a casual glance one might believe that a true septum was laid down. 
 That the spore is but 1 -celled can readily be demonstrated by clearing 
 it with a little salicylic acid. The variation in the size of 
 the pycnospores from various sources is seen from the 
 following measurements : 
 
 From host. — 6.0 to 8.6 by 2.4 to 4.1 /x, average 7.5 by 
 3-23 M- 
 
 Stems of Melilotus alba. — 5.6 to 10.0 by 2.4 to 4.0 /*, 
 average 7.82 by 3.1 1 ix. 
 
 Corn meal. — 5.4 to 8.2 by 2.4 to 3.4 //, average 6.49 by 
 2.79 p. 
 
 Irish potato cylinders. — 5.4 to 8.5 by 1.7 to 3.0 n, aver- 
 age 6.36 by 2.59 (j.. 
 Corn-meal agar. — 5.1 to 8.3 by 2.4 to 3.4 /x, average 6.78by 2.85 /x. 
 Rice. — 5.4 to 8.5 by 2.0 to 3.4 n, average 6.45 by 2.80 /jl. 
 It will be noted that the spores are relatively large in artificial cul- 
 tures on stems of Melilotus alba, and small on cooked Irish potato cyl- 
 inders. This difference has been found rather constant, and is prob- 
 ably associated with vigor of growth. Of all media tried stems of 
 M. alba were found exceedingly favorable for spore formation, while 
 cooked Irish potato cylinders were rather unfavorable. 
 
 <* %V 
 
 Fig. 3.— Diapor- 
 the phaseolo- 
 rum: A group of 
 pycnospores. 
 Note the oil 
 droplets and 
 variations i n 
 size and shape. 
 X500. 
 
482 
 
 Journal of Agricultural Research 
 
 Vol. XI, No. 10 
 
 Under favorable conditions, such as in a nutrient broth or beef agar, 
 the pycnospores germinate in about six to eight hours at room temper- 
 ature. Previous to germination the spores 
 become almost spherical and slightly en- 
 larged. Usually but one germ tube develops, 
 but occasionally two (fig. 4). One or both 
 of the oil globules may disappear at this 
 stage or remain until the germ tube is several 
 times the length of the spore. Growth is 
 rather slow at first, the hyphal growth attain- 
 ing a length compared with the diameter 
 of the spores as shown in figure 4 in 48 
 hours. During this time branching may oc- 
 cur, and septa rarely are formed till later. 
 
 The bodies called stylospores are frequently 
 found either alone or accompanying the 
 Fig. A.—Diaporthe phaseoiorum: Ger- pycnospores in pycnidia on the pods. They 
 
 minating pycnospores. X500. have never been fpund Qn the leayes> 
 
 They are long, slender, hyalin, nonseptate, straight or curved bodies 
 (fig. 5) borne on short, rather stout awl- 
 shaped simple hyalin stylosporophores (fig. 
 6). Figure 5 shows some stylosporo- 
 phores with stylospores attached. They 
 are produced but sparingly the early part 
 of the season or when the pycnospores 
 are most abundant. As the season advances 
 the stylospores increase in number and 
 appear to be associated, though not always, 
 with a saprophytic existence. Following 
 the death of the host and the gradual 
 disappearance of the pycnospores the stylo- 
 spores increase and finally become very 
 numerous. Specimens which bore practically no stylospores in the fall 
 
 bore them in increasing numbers throughout 
 the winter. There is a possibility from the 
 data at hand that the stylospores may be 
 associated with a lack of food supply. They 
 are produced in some culture media freely, 
 but not until after the food supply becomes 
 more or less exhausted. Although in cultures 
 fig. 6-Diaporihe phaseoiorum: a the pycnospores are usually exuded from the 
 
 group of stylosporophores. X500. , , . 
 
 beak in seven days, the stylospores have never 
 appeared sooner than 1 1 and generally not before 15 to 30 days after inocu- 
 lation. The average size of the winter crop of stylospores developed 
 
 Fig. 5. — Diaporthe phaseoiorum: Styl- 
 osporophores. (a) The stylosporo- 
 phore is attached to the stylospore. 
 (6) Stylospore not attached to the 
 stylosporophore. X 500. 
 
Dec. 3, 1917 
 
 Podblight of Lima Bean 
 
 483 
 
 under natural conditions is considerably larger than those produced in the 
 fall or in culture. Measurements made of the fall stylospores from the 
 host vary from 11.7 to 31.0 lx by 1.4 to 2.0 fx, average 22.83 by 1.73 M , 
 while those collected on February 7 varied from 20.6 to 54.4 /x by 1.38 
 to 2.4 ix, average 32.44 by 2.0 ll. They are produced in culture on rice, 
 corn meal, Irish potato cylinders, and stems of Melilotus alba and vary 
 in length from 11.7 to 54.4 li 
 and in width from 1.38 to 
 2.4 n. 
 
 The function of these bodies 
 is not known. Repeated at- 
 tempts have been made to 
 germinate them but without 
 success. Reddick (29) has 
 suggested the possibility that 
 they were paraphyses and 
 Saccardo (31, p. 264) regards 
 them as conidiophores. Von 
 Hohnel (23, p. 526), described 
 a new genus, Myxolibertiella, 
 to include those species hav- 
 ing stylospores and pycno- 
 spores. He first placed the 
 genus in the order Melanco- 
 niales but later in the order 
 Phomatales. Bubak (2) and 
 Diedicke (13) both regard 
 them as spores. The latter 
 
 investigator for Convenience FlG- l-—Diaporthe phaseolorum: Mature hyphae. Note the 
 , . 1 1 t->. ... septations, oil droplets, and granules. X500. 
 
 designated the Phoma-hke 
 
 spores by the Greek letter alpha (a) and the stylospores by beta (13). 
 Shear {32), like Bubak, Diedicke, and others, regards these bodies as 
 spores and designates them as scolecospores. 
 
 There is nothing peculiarly distinctive about the mycelium. It is 
 hyalin, frequently branched, and septate. It varies considerably in 
 width and is often supplied with enlargements which bear a slight re- 
 semblance to chlamydospores. When young the cells are supplied with 
 small protoplasmic granules. When the hyphae are old, the cells are 
 filled more or less with oil globules or droplets (fig. 7) . 
 
 ASCOGENOUS STAGE 
 
 Specimens of Lima beans consisting of stems and pods collected in 
 October, 1914, and January 1, 1915, at Vineland, N. J., were wintered 
 out at Washington, D. C, and covered over with dirt and leaves. These 
 
484 Journal of Agricultural Research vo1.xi.Ng. 10 
 
 specimens were kept under observation through the remainder of the 
 winter and spring months. Pycnidia and pycnospores were found from 
 time to time, as well as an increasing number of stylospores. On June 2 
 pods brought into the laboratory bore a considerable number of mature 
 perithecia of an ascomycete, which was identified as Diaporthe phaseo- 
 lorum (C. and E.) Sacc. The writer was away for five weeks previous to 
 June 2 so it is not certain just when the development of perithecia began, 
 and for that reason it was not possible to follow the complete course of 
 development. The perithecia are scattered and buried beneath the 
 surface, most of the beak protruding. Plate 43, D and F, shows dia- 
 grammatically the arrangement of the perithecia and relatively the 
 depth they are sunken below the surface. Whether or not the perithecia 
 are all formed in the cavities of old pycnidia has not been definitely de- 
 termined. The pycnidia are rather numerous 
 and often coalesce, and as a matter of chance a 
 perithecium might preempt a pycnidium and be 
 formed therein. It is believed, however, that 
 this is not the rule. The perithecia are not irregu- 
 lar in shape, as might be expected if they were 
 formed in old pycnidia; but they usually bear a 
 rather definite and constant form (PI. 43, F). 
 They also have separate and distinct walls which 
 Fig. s.-Diaporthe pkaseoio- differ f rom the i ayers f the pycnidium. In no 
 
 rum: (a) Asci and (6) asco- 
 
 spores. X500. case have the spores or stylospores ever been 
 
 found associated with a perithecium, although 
 these bodies were present on the host at the time. 
 
 The perithecia are slightly flattened, 158 to 355.5 m, average 251.9 n 
 in width and from 1 10.6 to 205.4 /*> average 166.5 M m depth. The beaks, 
 many times longer than wide, are usually curved or hooked (PI. 43, D), 
 rarely straight, tapering gradually to the end. The perithecium, circum- 
 scribed by a dark layer of cells, is lined within by a thin, hyalin layer 
 from which the asci arise. The perithecium is evidently almost or 
 completely matured before the beak is formed. Plate 43, D, shows 
 beaks just appearing through the epidermis. Sections through peri- 
 thecia with beaks in the incipient stage show the wall layers to be per- 
 fectly formed and many mature asci present. The asci (fig. 8, a) are 
 sessile, clavate or fusoid, 28.0 to 46.2 /x by 5.2 to 8.0 /z, numerous, 
 and contain eight spores arranged mostly biserially. The ascospores 
 are apparently bound together or embedded in a gelatinous mass, since 
 they are firmly held together even after the apparent disappearance of 
 the ascus wall. The ascospores, (fig. 8, b) measuring 6.4 to 12.0 by 2.3 
 to 4.0/i, are spindle-shaped, oblong-lanceolate, i-septate at or near the 
 center, slightly or not at all constricted, and contain two Ko four oil 
 droplets. 
 
Dec. 3,1917 Podblight of Lima Bean 485 
 
 The ascospores germinate readily in most any nutrient medium in 
 about six to eight hours by the development of a single germ tube from 
 one or both cells (fig. 9). The hyphae branch is 12 to 24 hours, and septa 
 are laid down at about the same time. Growth is very rapid. At ordi- 
 nary room temperature a colony from a single spore becomes visible 
 to the naked eye in about five days. The ascogenous stage has not been 
 produced in culture. The transfer of a colony from a single ascospore 
 resulted after 7 to 10 days in the production of the pyenidial stage on 
 the same culture media on which pyenidia were developed by a transfer 
 of pyenospores in the same length of time. In other words, there is 
 no difference between the growth of the ascogenous strains and pyenidial 
 strains on the same culture media. 
 
 INOCULATION EXPERIMENTS 
 
 The inoculation experiments on which the conclusions of the parasitism 
 of the organism have been drawn were made in the greenhouse. It was 
 found that Lima beans grow normally and produce fruit as well under 
 greenhouse conditions as in the open field. Opportunities 
 were therefore afforded to study the parasitism of this dis- 
 ease under controllable conditions and at any time of the 
 year most suited to the needs of the work. 
 
 Controls were held with each experiment, the number 
 and results of which are shown in Table I following a dis- fig. 9 .—Diap r- 
 cussion of the inoculation experiments. The pods unless lum.^Gemt 
 otherwise stated were inoculated by inserting spores and nating asco 
 hyphse into a wound made by a sterile needle. While 
 later evidence will show that under suitable conditions infection may 
 take place without wounding, the relative humidity of a greenhouse is 
 usually so low that spores smeared on the surface often fail to germi- 
 nate. It was necessary, therefore, to protect the spores from drying out 
 by inserting them under the epidermis or by other means. In most 
 cases it was impossible to protect the spores against drying, owing to 
 the fact that when a Lima bean plant is fruiting abundantly it is gener- 
 ally too large to confine in an infection cage, though this was done with 
 some smaller plants (see p. 486). 
 
 All inoculations were made from cultures grown on stems of Melilotus 
 alba. The age of the culture was not considered or found important 
 further than that the culture should be vigorous and fruiting abundantly. 
 Spores were exuded from the pyenidium in about seven days after the 
 culture was inoculated and continued to do so for 30 to 40 days there- 
 after or until the culture was dried. Spores were well suited for inocu- 
 lation purposes until the culture medium was dried out. 
 
 Inoculations were made with six different strains. By a strain is here 
 meant a difference in the source and not a difference in morphology or 
 
486 Journal of Agricultural Research voi.xi,No. 10 
 
 parasitology, since all were, found to be identical organisms. For the 
 sake of convenience in presentation each of the strains was given a sepa- 
 rate number as follows: Strain 286 was isolated on October 26, 1914, 
 from a Lima bean pod collected from a small patch at Vineland, N. J. 
 Stylospores and pycnospores were abundant. Strain 420 was isolated 
 from a Lima bean pod collected during the fall of 1914 by Mr. M. B. 
 Waite in Maryland. Stylospores, as well as pycnospores, were present 
 in abundance. Strain 447 was isolated from material collected on 
 January 1, 1915, from the same field as No. 286. Strain 567 is a reisola- 
 tion made on April 12, 191 5, from a pod that had been inoculated in the 
 greenhouse on March 13, 191 5, with strain 447. Pycnospores were 
 abundant, but there were no stylospores. Strain 598, as previously 
 stated, resulted from the isolation on June 4, 191 5, of a single ascospore 
 from a pod that had been wintered out at Washington, D. C. Strain 
 601 was isolated on August 7, 191 5, from leaves of Lima beans collected 
 at Cape May, N. J., on August 5, 191 5. 
 
 Other isolations of the same organism not used for inoculation pur- 
 poses have also been made for the purpose of identification and com- 
 parison. Frequent reisolations were made from inoculated pods, one 
 strain (567) of which was used to make inoculations. From specimens 
 inoculated with this strain the causal fungus was again recovered, thus 
 fulfilling the requirement of Koch's postulates, claimed necessary to 
 prove parasitism. 
 
 The first infection experiments were made on January 15, 191 5, when 
 20 nearly mature pods on six small plants were inoculated, 10 with 
 organism 286 and 10 with organism 420. After the pods were inoculated 
 by inserting spores and hyphse, the plants were sprayed with spores 
 suspended in water. They were then kept in a paper-covered infection 
 cage for 24 hours. On February 20, pycnidia were abundant on the 
 stems and on six pods inoculated with No. 286. Of those inoculated 
 with No. 420, five pods were dead, and pycnidia were already on four 
 and developed on one more after 10 days in a moist chamber. In this 
 case there were no leaf infections, though the stems, particularly the 
 lower parts, were covered with pycnidia, the pycnidia developing most 
 abundantly on the moribund stems and pods. 
 
 On March 13, 21 small plants were sprayed with organism 447. There 
 were only three small, immature pods in all and they were inoculated by 
 inserting spores and hyphae. These plants were inclosed for 48 hours 
 in paper-covered infection cages. Twelve days later the three pods were 
 drying up, and pycnidia were present on one and developed on the two 
 others after 7 days in a moist chamber. Here again no leaves were 
 infected, and the pods did not show typical infections. These results 
 were disappointing at the time, but it was discovered later that young 
 leaves and pods were difficult to infect, mature parts being much more 
 susceptible to the disease. 
 
Dec. 3,i9i7 Podblight of Lima Bean 487 
 
 On April 9, 1 91 5, twelve almost mature pods were inoculated by 
 inserting spores and hyphae in the usual way with organism 447. The 
 plants were too large to place in the infection cage. Seven days later 
 the tissue for half an inch or so around the point of infection was visibly 
 darker. Three days later some of the pods were withering, and pycnidia 
 began breaking through the epidermis % to % inch from the point of 
 inoculation. As long as the pods remain firm, the pycnidia form in 
 more or less concentric rings ; but concomitant with the withering of the 
 pods the fungus rapidly invades all the tissue, and pycnidia break through 
 the epidermis indiscriminately in a manner shown by Plate 42, C. Eight 
 of these pods finally became infected. 
 
 April 20, nine half-grown pods were sprayed with a suspension of 
 organism 447 in sterile water and kept in an infection cage for 24 hours. 
 These specimens were kept under observation until June 2, but no 
 infection had taken place. It is believed that the failure here is again 
 due to the relative immaturity of the pods. These results agree with 
 those of Pool and McKay (27), who were able to infect only mature 
 leaves of sugar beets with Phoma betae (Oud.) Fr. Apparently the writer 
 has succeeded in infecting young pods by wounding, but there is reason 
 for doubt, owing to the fact that a small wound frequently results in 
 serious injury and death of the pod, in which case the fungus quickly 
 invades it as a saprophyte. On April 23, spores of organism 447 were 
 inserted in five pods, and by June 2 pycnidia and spores were abundant 
 on three and a few days later on one more, one remaining sound. 
 
 On June 2, pods bearing perithecia which contained mature asci were 
 brought into the laboratory and thoroughly washed and disinfected in 
 mercuric chlorid. The perithecia were carefully picked out and macer- 
 ated in sterile water in a watch glass. Twenty-one nearly full-grown 
 pods were inoculated by inserting ascospores and bits of broken tissue 
 into wounds. Twelve days later thirteen pods showed unmistakable 
 evidence of the disease, and by June 2 1 all but four of the pods were well 
 covered with the fruiting bodies. The writer does not claim any proof 
 of the connection of the conidial and perfect stage from this experiment. 
 It is included here because it forms a link in the chain of experiments 
 performed with this organism. The results are justly open to the criti- 
 cism that spores and hyphae of the imperfect stage may have been 
 inserted, and it is not unlikely that such was the case. 
 
 On June 3, 191 5, fifteen pods were inoculated in the usual way with 
 organism 567. By June 14, pycnidia were abundantly produced over 
 an area an inch or more in diameter on ten pods, and on four others 
 by June 21. One pod alone remained sound. 
 
 On June 14, seventeen pods were inoculated with organism 598 
 (ascospore strain). In four days the tissue around the wound on some 
 pods began to darken, showing unmistakable and characteristic symptoms 
 of the disease. As the pods were killed by the fungus, they were gathered 
 
4 88 
 
 Journal of Agricultural Research 
 
 Vol. XI, No. 10 
 
 and all but one bore numerous pycnidia by August 9. Cultures from some 
 of these infected pods produced a growth characteristic of the pycnidial 
 stage. On the same date spores of organism 598 were smeared on the 
 surface of five nearly full-grown pods. There were no signs of infection 
 on June 2 1 , but by July 7 three showed evidence of infection, two remained 
 sound. Seven pods of various ages were sprayed with organism 598 and 
 confined in an infection cage for 30 hours. Pycnidia appeared on one of 
 these pods on June 21 and on two others on August 9 as well as on the 
 stems and some of the dead branches. The writer was away for sometime 
 previous to August 2, and it is not definitely known just how the disease 
 developed. It is probable, in view of the fact that the pods were dead 
 and studded all over with pycnidia, that they succumbed sometime 
 earlier. 
 
 On June 21 twenty-one pods were inoculated with organism 598 in the 
 usual way. One diseased pod was removed on June 29, eighteen on July 
 7, and one on August 17. One of the inoculated pods could not be found, 
 and was therefore regarded as not infected. There were ten checks held 
 for this experiment, one of which became infected and the only one of all 
 the checks (66) held at various times. 
 
 On August 17, ten pods were inoculated with organism 601, and by 
 September 3 six were well covered with pycnidia. Four remained sound. 
 Of 19 pods inoculated with 601 on September 7, ten showed typical in- 
 fection on September 25. Nine remained sound. 
 
 A summary of the results is given in Table I. 
 
 Table I. — Summary of inoculation experiments of Lima beans 
 
 Organism No. 
 
 Date of in- 
 oculation. 
 
 How inoculated. 
 
 Number in- 
 oculated. 
 
 Num- 
 ber in- 
 fected. 
 
 Num- 
 ber of 
 checks. 
 
 Num- 
 ber of 
 checks 
 
 in- 
 fected. 
 
 286. 
 
 420. 
 447- 
 
 447 
 
 447 
 
 447 ;■ 
 
 Fragments of peri- 
 thecia contain- 
 ing asci from 
 pods. 
 
 567 
 
 598 
 
 598 
 
 598. 
 598. 
 601. 
 601. 
 
 1915- 
 Jan. 15 
 
 ..do 
 
 Mar. 13 
 
 Apr. 9 
 
 Apr. 20 
 
 Apr. 23 
 
 June 2 
 
 June 3 
 June 14 
 ..do 
 
 ..do 
 
 June 2 1 
 Aug. 17 
 Sept. 7 
 
 By insertion of 
 spores in a wound. 
 
 do 
 
 Sprayed 
 
 Wounding 
 
 By wounding 
 
 Spraying 
 
 By wounding 
 
 do 
 
 ....do 
 
 ....do 
 
 Spores smeared on 
 pods. 
 
 Sprayed 
 
 By wounding , 
 
 ....do 
 
 ...do 
 
 10 pods. . . 
 
 ..do.... 
 
 21 plants 
 3 pods. . 
 12 pods. 
 9 pods. . 
 5 pods.. 
 21 pods. 
 
 15 pods. 
 17 pods. 
 5 pods. . 
 
 7 pods . . . 
 21 pods. . 
 10 pods . . 
 19 pods. . 
 
 4 
 17 
 
 14 
 
 16 
 
 3 
 
 3 
 
 20 
 6 
 
 10 
 
1 
 / 
 
 I 
 
 Dec. 3. 1917 Podblight of Lima Bean 489 
 
 PERPETUATION OF THE FUNGUS 
 
 The finding of mature asci of this fungus on June 2, 191 5, and on June 
 11, 19 1 6, and later on the dead vines and pods wintered out at Washing- 
 ton, D. C, and on similar material under field conditions at Vineland, N. J., 
 at about the same season of the year shows one way the fungus may live 
 through the winter. This method of perpetuation is, however, not the 
 only one. At intervals from October until June pycnospores were fre- 
 quently found, and the fungus was isolated at will from field material. 
 It is interesting to note in this connection that the pycnospores were 
 produced periodically during the winter season, depending upon weather 
 conditions. Following a period of a few days of relatively warm, rainy 
 weather, they could nearly always be found in more or less abundance. 
 For example, pycnospores were found on material examined on October 
 24, January 2, 14, and 21, February 7, March 6, April 14, and June 2. 
 Isolations were made on October 24, January 2, February 7, May 31, and 
 June 2. Similar results were obtained with Phomopsis vexans (Sacc. and 
 Syd.) Harter, the pycnospores of which were found in May and June on 
 wintered-out material. However, the same material examined in Decem- 
 ber, January, February, and April bore no pycnospores but the stylospores 
 appeared to be more numerous during the spring months. The appear- 
 ance of pycnospores was roughly coordinated with rainfall and tempera- 
 ture, since on May 22, following a period of rainy weather and relatively 
 high temperatures, they were found in great abundance. 
 
 It is likely also that the organism may live for a considerable time in 
 the seed. The writer has isolated it in pure culture from the interior of 
 both seeds and pods after surface disinfection in mercuric chlorid. From 
 these results it is apparent that the fungus invades all parts and it is not 
 unlikely that it is carried fron one season to the next on the seed. That 
 the fungus is not readily killed by drying is evident from the fact that it 
 was isolated from dried specimens kept in the laboratory for nine months. 
 It would probably survive such conditions even longer. These results 
 are supported by the earlier work of Halstead (17), who in 1901 called 
 attention to the fact that the seeds were invaded by the fungus and sug- 
 gested the possibility that it was carried through the season by that 
 
 means. 
 
 MODE OF INFECTION 
 
 It is not always easy to explain how a parasite gains entrance into its 
 host. It is safe to conclude that, if a fungus is parasitic, it may enter 
 through wounds, it being merely necessary to show that the host is 
 subject to wounding under natural conditions. Other types of penetra- 
 tion as, for example, through the unbroken epidermis or by way of the 
 stomata are not always easy to demonstrate. There is no doubt that 
 physical factors such as rainfall, temperature, humidity, and stomatal 
 movement all play an important r61e in the penetration of the epidermis 
 
49° Journal of Agricultural Research vol. xi, No. w 
 
 or stomata by certain parasites. In this connection it is interesting to 
 note the results of Pool and McKay (28), who have studied in some 
 detail the relation of stomatal movement to infection with Cercospora 
 beticola Sacc. These investigators showed that the germ tubes entered 
 only when the stomata were open, the movement of which was closely 
 associated with a relatively high humidity, warm temperature, and 
 light. Their results further showed that mature leaves were more readily 
 infected and possessed a pore opening almost twice as large as young 
 immature ones. The writer obtained similar results with the Lima bean 
 fungus. It was found, as already pointed out, that young immature 
 pods were not infected without wounding while mature pods could 
 easily be. This difference may be due at least in part to the difference 
 in the size of the stomatal aperture but more likely to the fact that im- 
 mature stomata do not open, thus preventing the entrance of the hyphae. 
 Under humid conditions the spores germinate and produce a germ tube 
 
 r^ 
 
 Fig. 10. — Diaporlhe pkaseolorum: Some of the germ tubes of the pycnospores penetrating the stomata two 
 days after the pods were sprayed, and other germinated spores lying alongside the stomata and growing 
 toward or away from the aperture. X 260. 
 
 20 or 30 times the length of the spore in 24 to 48 hours. This is rather 
 a feeble growth but the spores do not germinate readily in water, and 
 then only a small percentage at that. On pods that had been sprayed 
 with a spore suspension in water the germ tubes were found penetrating 
 the stomata as shown by figure 10. 
 
 It is evidently a matter of chance when a germ tube finds its way into a 
 stoma, since, if there was any attractive force, the germ tubes might be 
 expected to head that way, which is apparently not the case. The germ 
 tubes, as often observed by the writer and illustrated by figure 10, grow 
 just as frequently alongside or away from the stomata as toward them, 
 showing that there is no appreciable chemotactic influence. Entrance of 
 the stoma would be easy so far as relative size of germ tube and stom- 
 atal aperature are concerned. In fact, it would not be impossible and 
 it may actually happen that the spores fall directly through the pore 
 into the intercellular space beneath. Measurements of the pores showed 
 them to vary in length from 22 to 28 fx and in width from 5 to 7 /jl when 
 open, the germ tube of the spore in width from 2 to 3.5 /*, and the spore 
 itself from 6 to 8.6 n by 2.4 to 4.1 fi. Considering the relative size of 
 
Dec. 3, 1917 
 
 Podblight of Lima Bean 49 l 
 
 the stomatal pore and the spore of the fungus, it is quite reasonable to 
 conceive of an infection even during dry times, an event which actually 
 does sometimes take place. It must of course be admitted, and the ob- 
 served fact supports such a conclusion, that the number of infections by 
 a fungus of this type during dry weather is relatively small compared 
 with those during a period of rainfall or damp weather. Jones, Giddings, 
 and Lutman (23) pointed out that moist, damp weather was conducive 
 to the germination of the spores of Phytophthora infestans and made the 
 interesting observation that the germ tube penetrated the leaf of an 
 Irish potato through the epidermis as well as through the stomata. 
 Similar observations were made by Jones (24), who found that the germ 
 tube of Macrosporium solani E. and M. may penetrate the epidermis, as 
 well as the stomata of Irish potatoes. The writer found no instance of 
 penetration of Lima bean pods except through the stomata, and Pool 
 and McKay (28) make a similar statement for Cercospora beticola. 
 
 The inoculation experiments already described have shown that 
 infection takes place readily through wounds, perhaps made chiefly 
 by wind, and by means of pickers. In ordinary farm practice the pole 
 Lima bean is usually trained up several feet above the ground. Exposed 
 thus to the wind, the vines are whipped around and the pods wounded 
 by rubbing against the stems, poles, or wires on which they are trained. 
 A careful examination will readily show the bruised spots on pods ex- 
 posed to such conditions. Pickers also, in searching through the foliage, 
 may likewise brush or press the immature pods against stems or poles, 
 bruising them in a similar manner. The reddish, streaked appearance 
 in localized spots often observed on bean pods is characteristic of wounds 
 made by constant brushing or rubbing of the pod against a hard surface. 
 It is in such areas that infections are frequently found. 
 The relation of insects to infection has not been studied. 
 
 DISSEMINATION 
 
 The dissemination of the podblight of Lima beans must be considered 
 from two standpoints: (1) Over short distances, and (2) over long dis- 
 tances, as from one district or State to another. As to the first of these, 
 there are several ways in which the spores may be carried from plant 
 to plant or from one field to another. It is not unlikely that insects, 
 whether they feed on the pods or not, play an important part in the 
 distribution of the spores. During wet periods the spores are exuded 
 from the pycnidium, and any insect frequenting the diseased pods is 
 likely to carry the spores about on its body. Pickers, too, in searching 
 through the foliage for mature fruit, are likely also to distribute the 
 spores to unpicked pods, where, under favorable conditions, new infec- 
 tions will start. It is interesting to note in this connection that this 
 and certain diseases of other crops seldom become serious until about 
 picking time. There are two possible explanations for this. One is 
 
49 2 Journal of Agricultural Research voi.xi.no. » 
 
 that the host may be more susceptible at this age; and the other that 
 picking of the fruit, especially if the foliage is wet with dew or from 
 rain, results in a liberal distribution of the spores. 
 
 That strong winds may be responsible for severe outbreaks of this 
 and other diseases is evident from the fact that Cook (6, p. 517-518) 
 found a serious outbreak in a certain section of New Jersey previously 
 visited by a severe windstorm. It is, of course, natural to conclude, 
 as Cook assumed and the writer has frequently observed, that injury 
 to pods and foliage during such a storm would add to the chances of 
 infection. It is believed that the part played by the wind in dissemi- 
 nating fungus spores has been very greatly underestimated. It is a 
 well-known fact that the pollen of pine trees is carried long distances. 
 Dr. Albert Mann, of the Bureau of Plant Industry, found * a large 
 variety of fungus spores collected on a gelatin plate exposed for about 
 15 minutes at a distance of 1 mile above the surface of the earth. This 
 readily shows that spores are freely air-borne over long distances, and 
 this fact alone may account for the serious outbreak of a disease in 
 isolated sections. 
 
 Owing to the fact that the causal organism can readily be isolated 
 from the seed, it is likely that this affords the principal means of dis- 
 tributing it over long distances. Fruiting bodies have also been found 
 on infected seed. This shows the great necessity of saving seed only 
 from fields where this disease does not occur. 
 
 CULTURAL CHARACTERISTICS OF THE FUNGUS 
 
 The fungus produces hyphse on nearly all of the culture media in 
 ordinary use. The pycnidia, on the other hand, are rarely produced 
 on agars, but freely on media containing starch, such as rice or corn 
 meal. A study of the growth and production of fruiting bodies was 
 made on the following media: String-bean agar, Irish potato agar, beef 
 agar, corn-meal agar, cooked rice, cooked corn meal, potato cylinders, 
 and stems of Melilotus alba. These media were selected because they 
 are in common use in the laboratory of Cotton, Truck, and Forage Crop 
 disease investigations, and because some (corn meal and rice) were 
 known to be particularly favorable for the growth of other very similar 
 organisms, thus affording an opportunity for comparison. Other media 
 might have served the same purpose. 
 
 The experiment was carried out by inoculating five tubes (100 c. c. 
 flasks, in case of corn meal) with spores from a 10-day-old culture on 
 stems of Melilotus alba. After inoculation the tubes were kept in the 
 light and exposed to the temperature of the laboratory room (2i°-25°C). 
 
 String-bean agar. — This on the whole has been a poor medium for this fungus. 
 Growth of hyphae started promptly and was visible in two days in slanted tubes of 
 
 1 Unpublished data. 
 
Dec. 3, 1917 Podblight of Lima Bean 493 
 
 the medium. At the end of 9 days three dark bodies, which were at that time thought 
 to be p}'cnidia, and which probably were sterile ones, were developed. These cul- 
 tures were kept under observation for 72 days, and no more pycnidia and no spores 
 were found. 
 
 Irish potato agar. — There was a visible vegetative growth in two days which 
 continued to increase for about one week. A few scattered pycnidia appeared in all 
 tubes but one. These were kept under observation for 72 days, and no spores or 
 stylospores were ever produced. 
 
 BEEP agar. — A rapid vegetative growth during the first seven days of growth. 
 Thereafter the hyphae became fluffy and cottony. No pycnidia were formed in any 
 of the tubes during the whole course of the experiment. 
 
 Corn-meal agar. — Growth was not visible in two days, but in four days the 
 hyphae had spread over an area J4 inch in diameter. In six days dark specks appeared 
 which later developed into mature pycnidia. At the end of 13 days the slimy white, 
 creamy masses began to exude from the pycnidia. These cultures were frequently 
 examined thereafter for stylospores, but none were found until after 34 days, when 
 they appeared in great abundance. When stylospores and pycnospores are both 
 produced, the former always appear after, and never before, the production of the 
 latter. 
 
 Cooked rice. — Rice has proved to be a good medium for the growth of this organ- 
 ism. Hyphae were conspicuous and pycnidia began to form in 4 days. From that 
 time the pycnidia increased in number and spores began to exude in 7 days. A 
 heavy, black stromatic mass, in which the pycnidia were embedded, was formed. 
 As the medium dried out, these stromatic masses were left as conspicuous domelike 
 projections over the surface of the medium. The pycnospores were exuded in slimy, 
 yellowish, viscid masses in such quantities as to flow over the top of the stroma and 
 mostly to cover the surface of the medium. Stylospores were found sparingly in 22 
 days and abundantly in 34 days. 
 
 Cooked corn meal. — Corn meal was found to be one of the best media for this and 
 other closely related fungi, such as Phomopsis vexans, Plenodomus destruens, and 
 Diaporthe batatatis. In 5 days the hyphae covered most of the surface of the corn 
 meal in 100-c. c. flasks, and pycnidia began to form. The pycnidia were produced 
 in great numbers in a more or less well-developed stroma which bore characteristics 
 similar to those on rice. The small slimy droplets containing pycnospores were 
 first noticed at the end of 13 days. The slimy exudate increased in amount until 
 the droplets became quite large and flowed over the sides of the stroma, eventually 
 covering nearly the whole surface of the medium. The stroma and pycnidia were 
 dark on top and somewhat raised above the surface of the medium. As the corn meal 
 dried out, the surface became more or less crusty and rugose. Stylospores were found 
 for the first time at the end of 22 days. 
 
 Potato cylinders. — On this medium abundant mycelium was produced. A 
 cottony, white growth covered one-third of the potato slant in two days and com- 
 pletely in four days. In five days incipient pycnidia were seen which later developed 
 into a more or less stromatic mass. These masses were at first whitish on top, but 
 later turned darker. In 22 days pycnospores were exuding slightly, and stylospores 
 were present. At the end of 34 days the stylospores had become more abundant than 
 on any other medium; the pycnospores, on the other hand, were relatively few. 
 
 Stems oe melilotus alba. — This medium, everything considered, is the best 
 of any so far used. It gives a maximum amount of spores with a minimum of mycelium. 
 There were no signs of growth in two days, but in five days numerous pycnidia began 
 to form, and these soon became conspicuous as black specks studding the surface of 
 the stems. The beaks were comparatively long and conspicuous. In one week 
 under optimum conditions the viscid, creamy droplets in which the spores are em- 
 
494 Journal of Agricultural Research vol. xi, No. 10 
 
 bedded were noticeable on the end of the beak. These droplets gradually enlarged 
 and finally overflowed the pycnidium, coalescing with similar droplets from other 
 pycnidia and thereby forming an almost continuous liquid covering of the substratum. 
 A stroma was not formed on stems of Melilotus alba. In this particular experiment 
 no stylospores were formed. In other cultures, however, of the same organism on 
 M. alba stems a few stylospores were found. 
 
 EFFECT OF LIGHT ON GROWTH 
 
 The literature is full of the records of experiments on the effect of light 
 on growth and development of chlorophyll-bearing plants, as well as 
 fungi. It would be useless to review all or any considerable part of these 
 records here. It is evident, however, from a perusal of some of these 
 publications that no general, sweeping conclusions can be drawn that 
 will apply to all fungi alike. Recent results, on the other hand, show that 
 under similar conditions individual fungi may be expected to respond 
 differently. In support of this last statement, reference may be made 
 to the results of Coons (10), who found that light was a determining factor 
 in the production of pycnidia with Plenodomus fuscomaculans (Sacc.) 
 Coons. The writer (19), on the other hand, working with a different 
 species of Plenodomus, P. destruens Harter, found that the absence of 
 light did not inhibit the production of pycnidia. That the absence of 
 light very greatly inhibited, but did not entirely prevent, the production 
 of pycnidia by Diaporihe batatatis, was shown by Harter and Field (21) 
 in another experiment. The writer has obtained similar results, as yet 
 unpublished, with other fungi. 
 
 The influence of light on growth and production of fruiting bodies of 
 the pycnidial stage of Diaporihe phaseolorum was determined by inocu- 
 lating stems of Melilotus alba in test tubes and exposing one-half of the 
 set to light and excluding the light from the other tubes by wrapping 
 with black paper, which was found by previous test to prqhibit the 
 passage of light. The tubes were all tightly plugged with cotton. The 
 cotton plugs alone were left exposed, so as to permit aeration. The two 
 series were then placed on a table in the center of the laboratory room 
 and kept under observation for 25 days. The cultures began to dry up 
 about that time and development was accordingly arrested. The tubes 
 kept in the dark were examined for a few minutes every few days for 
 the purpose of taking notes and were for that length of time exposed 
 to the light. While this brief period can not be regarded as having 
 no influence on the results, exposure to the light for such a short time 
 would certainly have no marked effect. The growths of mycelium in 
 the cultures in the light and those in the dark were about the same at 
 the end of three days, but from that time the mycelial growth in 
 the dark was relatively more abundant and more cottony. In tubes 
 in the light, on the other hand, there was, as has always been, a minimum 
 of mycelium on stems of Melilotus alba. At the end of six days pycnidia 
 
Dec. 3, 1917 
 
 Podblight of Lima Bean 495 
 
 were forming in large numbers in all tnbes in the hght and m smaller 
 Zmbers in all those in the dark. The ratio between the number of 
 Turn a formed in the light and in the dark remained nearly constant 
 the fruiting bodies forming in all tubes, but they were better developed 
 n the light In one week spores began to exude from the pycmdia 
 both in the light and in the dark. From this time spores continued 
 to be exuded, but here again the amount of the exudate was relatively 
 less in the dark. Toward the close of the experiment, however, there 
 appeared to be a relative increase in the exudate from the pycmdia 
 formed in tubes in the dark. But one conclusion can be d™™*"" 
 these results, that while the dark retards the production of fruit ng 
 bodies and accelerates vegetative growth, it is not as marked as the 
 results obtained with other fungi by the writer and others. 
 
 INFLUENCE OF TEMPERATURE ON GROWTH 
 
 To determine the influence of temperature on growth 12 (No 2, 3, 5. 
 6 8 q .0 15 17, 18, .9, and 20) of the 20 chambers ,n an Altmann 
 incubator were used. Tubes of cooked rice were inoculated with spores 
 oTthe nngus and immediately placed in the chambers, where they were 
 kept exposed to the respective temperatures for a period of 27 days, at 
 thfendTwhich time growth had practically stopped. No growth took 
 place in chambers 2 (4.1° C). 3 (6..° C), 19 (35-4° O. and 20 (38 8 O 
 and but a scant growth in 18 (32.9° C). The optimum growth was 
 ana DU , , 5 , <,on.nHi7(an l, C) The growth of hypha: 
 found in chambers 1 5 (26.5° C.) and 17 U0.5 <-.)■ s ^ 
 
 decreased relatively with the increa* and decrease of the temperature 
 
 from these limits of the optimum. In chambers 9 (17.4 C .), 10 (19.4 
 from these > v ^ stromatlc f 
 
 pseudoparenchymo^ tissue were produced which macroscopically looked 
 hke pyenidia. A careful examination of these bodies, however showed 
 hat'while they bore some resemblance to pyenid, 1 ,n< , cavity had b 
 produced within them and no pyenospores were found. The cause or 
 tto was later found to be due to a lack of sufficient aeration, resulting 
 probably in a reduced oxygen supply. A subsequent expenmen, in 
 which cultures of the organism on cooked rice were exposed in a chamber 
 ta^hfchm^rf the oxygen had been removed by a mixture of pyrogalhc 
 acid audllm hvdroxfd showed that while mycelial growth continued 
 under such conditions the production of fruiting bodies was inhibited. 
 In" add ta o tte f act that'the cultures in the Altmann chambers were 
 tent in the dark, the absence of oxygen, which in itself was found as 
 aheady Sown, to reduce somewhat the production of «b^». 
 evidentlv accounted for the failure to produce pyenidia in the cited cases. 
 The growth of the podblight fungus in artificial cultures is schemati- 
 cally shown in figure n. 
 
496 
 
 Journal of Agricultural Research 
 
 Vol. XI, No. 10 
 
 /O /S 2Q PS SO .3 6- 
 
 4P 
 
 INFLUENCE OF TOXIC AGENTS ON GERMINATION OF SPORES 
 
 Perhaps no chemical substance has been more generally studied in its 
 relation to the growth of plants than copper salts. This is perhaps due 
 to its well-known efficiency as a fungicide, following the discovery of which 
 a large amount of work was undertaken to determine the concentration 
 of copper salts necessary to prevent germination or bring about the death 
 of the spores of a great variety of fungi. Much of this work was done 
 with saprophytes, and consequently has little more than a scientific 
 interest, while the practical side of the problem was evident from the fact 
 that the toxicity to germinating spores of many well-known parasites was 
 established. Because of the various uses that chemical substances may 
 be put to, such as antiseptics, disinfectants, etc., the toxic action of many 
 
 salts, metals, and acids, 
 alone and in combina- 
 tion, on flowering 
 plants and fungus 
 spores have been ex- 
 tensively studied, and 
 as a result an extensive 
 literature on the sub- 
 ject has accumulated. 
 As might be expected, 
 the results have been 
 very variable and often 
 contradictory. In gen- 
 eral, however, all in- 
 vestigations have shown that copper salts, among other chemical 
 substances, are exceedingly toxic to all germinating spores and to the 
 seedlings of flowering plants. Coupin (11) has shown by experiments 
 that 1 part of copper sulphate to 700,000,000 parts of water is suffi- 
 cient to retard the root growth of wheat seedlings, and the writer has 
 proved (18) that a small amount of a single alkali salt, such as 
 sodium chlorid, sodium carbonate, and magnesium sulphate in water 
 cultures, inhibits the growth of the same plant. Heald (22) found 
 1/51,200 and 1/25,600 gram-molecule per liter of copper sulphate to be 
 toxic to Pisum sativum and Zea mays, respectively. According to Moore 
 and Kellerman (26) 1 part of copper sulphate to 500,000 parts of water 
 killed Closterium moniliferum in 4 days and 1 part of copper sulphate to 
 3,000,000 parts of water killed Anabaena flos-aqwz in three days. 
 Uroglena americana was even more sensitive and was killed in 16 hours 
 in 1 part of copper sulphate to 10,000,000 parts of water. 
 
 These few references show the extreme sensitiveness of some of the 
 flowering plants and some algse to copper salts, and at the same time the 
 variability in their resistance. Although not strictly comparable with 
 
 Fig. i 1 —Diaporthe phaseolorum: Graphic representation of growth on 
 cooked rice at different temperatures. 
 
Dec. 3, 1917 Podblight of Lima Bean 497 
 
 flowering plants, because of the different methods or experimentation 
 and standards of measurement, the spores of fungi show equally great 
 variability and sensibility to copper salts and other toxic substances. 
 Clark (3) found that Penicillium glaucum was injured by copper sulphate 
 at a concentration somewhere between N 1 2,048 and N '/ 1,024, while 
 Aspergillus flavus would endure a concentration of N/512 to NI256, two 
 to four times as great. Crandall (12), on the other hand, showed that 
 the apple scab fungus is quite resistant to copper sulphate, the spores 
 being only slightly retarded in germination in a solution of 1 to 100,000, 
 while a concentration of about 1 to 10,000 was necessary to entirely 
 prevent germination. At this latter concentration some of the common 
 molds, however, grew quite well. Temperature was found by Brooks 
 (1) to influence the percentage of germination of the spores of certain 
 fungi in the same concentration of a toxic substance. By an extensive 
 series of experiments he found that Penicillium glaucum gave medium 
 growth in N/128 copper sulphate at 20 C, Monilia fructigena did not 
 germinate in N/16 copper sulphate at 25 to 30 C, but at 15 C. 12 per 
 cent, at io° C. 30 per cent, and at 5 C. 49 per cent. 
 
 No attempt has been made to review all the extensive literature on the 
 effect of toxic substances on plant growth. The results of the work of 
 the different investigators are very conflicting, due, perhaps in part at 
 least, to the different methods of experimentation employed and the 
 different standards of measurement. 
 
 During the time the podblight fungus of Lima bean has been under 
 investigation many interesting observations have been made from time 
 to time on the effect of certain chemical substances commonly used in the 
 laboratory on the germination of the spores. The use of a solution of 
 mercuric chlorid for disinfecting field specimens preparatory to isolating 
 the fungus led to a study of the strength of this solution necessary to kill 
 the spores ,n a given time. A similar study was made with formaldehyde 
 because of its possible use in disinfecting infected seed, and with copper 
 sulphate, a well-known constituent of Bordeaux mixture, sometime rec- 
 ommended as a spray in controlling the disease in the field. 
 
 METHOD OF EXPERIMENTATION 
 
 Preliminary experiments showed that the use of Van Tieghem cells 
 to determine the toxic limit for the germination of the spores was ex- 
 ceedingly unreliable and was consequently abandoned. Although this 
 method has been generally used, it is likely that many of the conflicting 
 results may be attributed to it. In fact, the writer found that each 
 test in Van Tieghem cells might be expected to give different results, 
 probably owing to the variation in vapor pressure or to some slight 
 accident overlooked, unexpected, and unaccounted for. After con- 
 sidering the different methods that might be used, the writer concluded 
 
498 Journal of Agricultural Research voLxi.no. 10 
 
 to employ the method frequently used in medical bacteriology to de- 
 termine the germicidal value of certain chemicals. This takes more 
 into account the actual death of the organism rather than the strength 
 that inhibits growth. It seems that the same method should apply 
 equally well to fungi and is easier of determination and more reliable, 
 since the length of time the organism is exposed plays an important 
 part also, and can be more accurately gauged. It is a well-known fact 
 that a fungicide may merely inhibit growth when, the spores are exposed 
 to it for a certain length of time but may kill them on prolonged ex- 
 posure. In these experiments the time the spores were exposed to the 
 fungicide was relatively short, the concentration of the solution being 
 proportionately strong to produce death in a comparatively short time. 
 This method eliminated the necessity of taking into account, among 
 other things, the temperature which Brooks (1) found to play such an 
 important part in germination of the spores of some fungi. 
 
 The pycnospores of Diaporthe phaseolorum were found to grow well in 
 beef agar + 15 on Fuller's scale; and therefore this medium was used in 
 which to test germination. The spores from a culture on Melilotus alba 
 were transferred by a small platinum loop to a solution of the fungicide 
 of the desired concentration and mixed by a wide circularly swinging 
 motion of the arm with the plugged end up, or by vigorous shaking. At 
 the end of one minute, two minutes, and every two minutes thereafter 
 up to the suspected limit of endurance, a loopful of the mixture was 
 transferred to a tube of liquefied agar and, after mixing by shaking, a plate 
 was poured. Control plates were made by transferring a loopful of 
 spores from the same test culture to a sterile water tube and a loopful 
 from this tube to a tube of liquefied agar. This method was followed 
 with each of the fungicides and repeated as often as necessary to establish 
 the toxic limit. The plates were kept at room temperature in the 
 laboratory. The colonies in the control plates appeared in about two 
 days. Notes were taken from time to time for seven days, after which 
 they were thrown out, for, if growth took place at all, it would do so 
 before that time. 
 
 RESULTS OF EXPERIMENTS 
 
 The results of these experiments are given in Table II. 
 
 The mercuric-chlorid and copper-sulphate solutions were made in 
 advance and kept corked in the ice box until needed. Because of the 
 instability of the formaldehyde, the solutions were prepared just before 
 using and in such a way that the fractional parts given in Table II are 
 based on a hypothetical formaldehyde solution of a strength of 100 per 
 cent. 
 
Dec. 3, 191 7 
 
 Podblight of Lima Bean 
 
 499 
 
 Table II. — The results of exposing pycnospores of Diaporthe phaseolorum to different 
 strengths of mercuric chlorid, copper sulphate, and formaldehyde for different lengths of 
 time 
 
 Time of 
 
 Copper sulphate. 
 
 Formaldehyde. 
 
 Mercuric chlorid. 
 
 exposure. 
 
 NI12.5 
 
 NI31.25 
 
 NI62.S 
 
 1:100 
 
 1:250 
 
 1:500 
 
 NI13S 
 
 Nli,3so 
 
 N 12,700 
 
 NI4.050 
 
 Minutes. 
 I 
 2 
 
 4 
 
 a 100 
 
 35 
 5 
 
 100 
 65 
 
 55 
 25 
 15 
 10 
 
 5 
 2 
 
 100 
 90 
 
 75 
 
 
 85 
 
 30 
 
 
 
 100 
 100 
 
 85 
 
 10 
 
 
 
 
 O 
 
 O 
 O 
 
 5° 
 O 
 
 IOO 
 IOO 
 
 75 
 5° 
 
 6 
 
 
 
 
 8 
 
 75 
 
 
 
 
 
 
 10 
 
 
 
 
 
 
 
 12 
 
 45 
 
 
 
 
 
 
 
 
 14 
 16 
 
 
 
 
 
 
 
 
 20 
 
 
 
 
 
 
 
 
 18 
 
 
 
 
 
 
 
 
 
 
 20 
 
 
 
 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 a In this and corresponding columns are given the percentage of germination as estimated at the close of 
 the experiment. Water controls were made in every case in which the germination was arbitrarily placed 
 at 100 per cent. These plates, together with those poured from a 1 minute exposure, served as controls for 
 comparison in estimating percentages of germination. 
 
 Reference to the table will show that the spores were nearly all killed 
 in four minutes in a N/12.5 copper-sulphate solution, and in 14 minutes 
 in a N/31.25, and in 20 minutes in a N/62.5 solution. It is interesting 
 to compare these results with those of Clark (3), who found that the 
 spores of several fungi exposed in a copper-sulphate solution were killed 
 or injured as follows: Aspergillus flavus (?) Link N/4 and greatly injured 
 in N/256, Sterigmatocystis niger Von Tieghem N/8 and greatly injured in 
 N/128, Oedocephalum albidum Sacc. N/64 an d greatly injured at N/256, 
 Botrytis vulgaris Fr. greatly injured at N/128, and Penicillium glaucum 
 Link N/i and greatly injured at N/256. As will be seen, there is a wide 
 difference between the strength of the solution necessary to kill and that 
 required to injure greatly the spores. The writer found that none of the 
 spores exposed for one minute to a solution of mercuric chlorid at a con- 
 centration of N/135 an d N/1,350 would germinate, and that only 50 per 
 cent germinated when exposed for the same length of time in a concen- 
 tration of N/3,700. At the greater dilution, N/4,050, 50 per cent germi- 
 nated after an exposure for six minutes. Clark (3) found that spores of 
 A. flavus, S. niger, P. glaucum were killed in a N/ 4,096 mercuric chlorid, 
 O. albidum in a N/ 16,384, and B. vulgaris in a N/ 65, 536. The spores of 
 these fungi were killed in the solutions of the toxic substance more dilute 
 than that required to kill the pycnospores of Diaporthe phaseolorum, but 
 they were likewise subjected to the solution for a longer time. In 
 another work Clark (4) has shown that Rhizopus nigricans was killed in 
 75 seconds in a solution of mercuric chlorid 1 to 3,700. 
 
 Formaldehyde was found to be very toxic to the spores, only 10 per 
 cent germinating after a six-minute exposure in a i-to-500 solution, and 
 
500 Journal of Agricultural Research vol. xi, no. io 
 
 30 per cent after a two-minute exposure in a i-to-250 solution, and 
 
 only 75 per cent in a 1 -to- 100 solution after one minute. Clark (3) 
 
 found that 1 part by weight in 273,066 parts of water permitted the 
 
 germination of but 10 per cent of the spores of Oedocephalum albidum 
 
 after 1 1 hours' exposure, as compared with 95 per cent after four hours 
 
 in the controls, and greatly injured the mycelial development. 
 
 As regards the results, it should be stated that in all cases where the 
 
 spores were not killed the germination was very much retarded. The 
 
 colonies in the controls became visible to the naked eye in about 48 
 
 hours. A long exposure in a weak solution retarded germination to 
 
 about the same^ degree that a short exposure in a correspondingly more 
 
 concentrated solution. That the germination of Lima bean seeds is not 
 
 injured by exposure for 10 minutes in the strongest solutions of mercuric 
 
 chlorid, formaldehyde, and copper sulphate to which the spores were 
 
 subjected is shown by a series of experiments discussed under "Control 
 
 measures." 
 
 CONTROL MEASURES 
 
 Halsted (17) was the first, and probably the only one, who has ever 
 done any experimental work on the control of the podblight of Lima 
 beans. In these experiments spraying the dwarf Limas with Bordeaux 
 mixture or soda-Bordeaux mixture yielded very favorable results. 
 Although he evidently did not spray the pole Limas, he suggests that the 
 disease could be checked by so doing. It is now known that the fungus 
 attacks the leaves before the pods are set, and it is likely the pods become 
 infected from them. In view of this fact, the first spraying should be done 
 when the plants are 1 or 2 feet high, and should be repeated often enough 
 to keep the foliage covered. 
 
 From badly infected pods the fungus invades the seed, where it may 
 live for a considerable time. Infected seed are usually dark, shriveled, 
 and immature. They probably would not grow, and, if they did, would 
 give weak, sickly plants. Only plump, bright seed should be planted. 
 Sound seed may be disinfected to kill the adhering spores by submerging 
 for 5 or 10 minutes in a solution of mercuric chlorid (1 : 1,000), formalde- 
 hyde (1:100), or copper sulphate (1:100), after which they should be 
 rinsed in water. 
 
 A series of experiments has shown that germination was not injured by 
 such a treatment. Healthy Lima bean seeds were sorted out; a part 
 were treated for 5 minutes and a part for 10 minutes. After rinsing in 
 sterile water, they were laid between moistened filter paper and ger- 
 minated. The results showed that seeds exposed for 10 minutes gave 
 just as good germination as those soaked in sterile water for the same 
 length of time. It should be mentioned in this connection that those 
 soaked in sterile water were all more or less covered with saprophytic 
 molds, while those treated with mercuric chlorid, formaldehyde, and 
 copper sulphate were comparativelv free. 
 
Dec. 3 , 1917 Podblight of Lima Bean 501 
 
 SUMMARY 
 
 (1) The podblight of Lima beans is probably indigenous to the United 
 States. 
 
 (2) It was first recognized by Halsted in New Jersey in 1891, and has 
 since been found in the States of Virginia, Maryland, Connecticut, and 
 North Carolina. 
 
 (3) The disease forms circular brown spots on the leaves, and large 
 unsightly spots on the nearly mature pods and on the stems. Numerous 
 pycnidia are produced in the diseased areas. 
 
 (4) The loss, which in some fields is very large, is confined mostly to 
 the pods. 
 
 (5) The Lima bean podblight fungus has been known as Phoma 
 subcircinata E. and E. almost since its first discovery, although in a few 
 cases it has been referred to as a species of Phyllosticta. 
 
 (6) The writer isolated single ascospores of a Diaporthe found on 
 specimens wintered out and made inoculations with pure cultures of 
 such strains which produced a disease identical with that produced by 
 inoculations with pure cultures of a pycnospore strain. In 1 892 Diaporthe 
 phaseolorum was completely described, which agrees with the ascogenous 
 fungus the writer has been studying. The fungus causing the disease 
 is therefore referred to as Diaporthe phaseolorum (C. and E.) Sacc. 
 
 (7) The hyphae course through and among the parenchymous cells 
 of the pod, forming pycnidia just under the epidermis at points where 
 the bast cells are more or less separated. 
 
 (8) Stylospores are abundantly found on field material and readily 
 produced in culture on some media. The pycnospore stage belongs to 
 the form genus Phomopsis and not to Phoma. 
 
 (9) The disease is readily produced by artificial inoculation with spores 
 from both the pycnidial and ascogenous strains. 
 
 (10) Wounding of the pod is not necessary for infection, as the germ 
 tubes may enter through the stomata. 
 
 (11) The fungus is carried through from one season to the next, either 
 by periodic production of pycnospores and the persistence of hyphae or 
 by the production of an ascospore stage. 
 
 (12) The disease may be carried on the seed and spread by such 
 agencies as wind, by the process of picking, and possibly by means of 
 insects. 
 
 (13) The fungus fruits well on stems of Melilotus alba, on rice, corn 
 meal, and other starch media, and but poorly or not at all on the agars 
 tried with the exception of corn-meal agar. 
 
 (14) Cultures in the dark fruited more slowly than those in the light 
 and had increased vegetative growth. 
 
 (15) No growth takes place at a temperature below 6.i° nor higher 
 than 35. 4 C. during a period of 27 days. 
 
502 Journal of Agricultural Research vol. xi. No. 10 
 
 (16) Dilute solutions of formaldehyde, copper sulphate, and mercuric 
 chlorid are toxic to the spores. The germination of the spores is greatly 
 retarded when exposed to a solution too dilute to kill them in a given 
 time. 
 
 (17) To control the podblight it is recommended that clean seed be 
 selected. They should then be disinfected in mercuric chlorid, formalin, 
 or copper sulphate. The plants should be sprayed in the field the first 
 time when they are 1 to 2 feet tall, and often enough thereafter to keep 
 the foliage covered. 
 
 LITERATURE CITED. 
 
 (1) Brooks, Charles. 
 
 1906. temperature and toxic action. In Bot. Gaz., v. 42, no. 5, p. 359-375, 
 12 fig. Literature cited, p. 375. 
 
 (2) Bubak, Franz, and Kabat, J. E. 
 
 1905. VIERTER BEITRAG ZUR PILZFLORA VON THIOL. In OsteiT. Bot. Ztschr., 
 
 Jahrg. 55, no. 2, p. 73-79. (Continued article.) 
 
 (3) Clark, J. F. 
 
 1899. ON THE TOXIC EFFECT OF DELETERIOUS AGENTS ON THE GERMINATION 
 AND DEVELOPMENT OF CERTAIN FILAMENTOUS FUNGI. In Bot. Gaz., 
 v. 28, no. 5, p. 289-327; no. 6, p. 378-404, 10 diagr. Bibliography, 
 p. 402-404. 
 (4) 
 
 19OI. ON THE TOXIC VALUE OF MERCURIC CHLORIDE AND ITS DOUBLE SALTS. In 
 
 Jour. Phys. Chem., v. 5, no. 5, p. 289-316, 7 fig. 
 
 (5) Clinton, G. P. 
 
 1906. REPORT OF THE BOTANIST. NOTES ON FUNGUS DISEASES, ETC., FOR 
 
 1905. In Conn. Agr. Exp. Sta., 29th Ann. Rpt., [i904]/o5, p. 263-277. 
 
 (6) Cook, M. T. 
 
 1913. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 33d 
 
 Ann. Rpt., [i9ii]/i2, p. 509-527. 
 
 (7) 
 
 1914. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 34th 
 
 Ann. Rpt., [i9i2]/i3, p. 793~8i7> 13 fi g- 
 (8) 
 
 191 5. REPORT OF THE PLANT PATHOLOGIST. In N. J. Agr. Exp. Sta., 35th 
 
 Ann. Rpt., [i9i3]/i4, p. 467-476. 
 (9) Cooke, M. C, and Ellis, J. B. 
 
 1878. new jersey fungi. In Grevillea, v. 6, no. 39, p. 81-96. (Continued 
 article.) 
 (10) Coons, G. H. 
 
 1916. FACTORS INVOLVED IN THE GROWTH AND THE PYCNIDIUM FORMATION OF 
 
 plEnodomus fuscomaculans. In Jour. Agr. Research, v. 5, no. 16, 
 p. 713-769. Literature cited, p. 766-769. 
 (n) Coupin, H. 
 
 19OI. SUR LA SENSIBIEIT6 DES v£g6TAUX SUPERIEURS A DES DOSES TRES 
 FAIBLES DE substances ToxiquES. In Compt. Rend. Acad. Sci. 
 [Paris], t. 132, no. 10, p. 645-647. 
 
 (12) Crandall, C. S. 
 
 1909. bordeaux mixture. 111. Agr. Exp. Sta. Bui. 135, p. 199-296, 8 fig. 
 
 (13) Diedicke, H. 
 
 1911. die gattung phomopsis. In Ann. Mycol., Jahrg. 9, no. 1, p. 8-35, 3 pi. 
 
Dec. 3. 191 7 Podblight of Lima Bean 503 
 
 14) Ellis, J. B., and Everhart, B. M. 
 
 1 892. THE NORTH AMERICAN PYRENOMYCETES. 793 p., 41 pi. Newfield, N. J. 
 
 15) 
 
 1894. NEW SPECIES OF NORTH AMERICAN FUNGI FROM VARIOUS LOCALITIES. 
 
 In Proc. Acad. Nat. Sci. Phila., 1893, p. 128-172. 
 16) Halsted, B. D. 
 
 1892. lima bean diseases. In N. J. Agr. Exp. Sta., 12th Ann. Rpt, 1891, 
 p. 287. 
 
 17) 
 
 1901. bean diseases and THEIR remedies. In N. J. Agr. Exp. Sta. Bui. 151, 
 28 p., 9 fig., 4 pi. 
 18) Harter, L. L. 
 
 1905. THE VARIABHJTY OF WHEAT VARIETIES IN RESISTANCE TO TOXIC SALTS. 
 
 U. S. Dept. Agr. Bur. Plant Indus. Bui. 79, 48 p. Bibliography, 
 p. 47-48. 
 19)- 
 
 20) 
 
 1913. the foot-rot OF THE SWEET potato. In Jour. Agr. Research, v. 1, 
 no. 3, p. 251-274, 1 fig., pi. 23-28. 
 
 1914. FRUIT-ROT, LEAF-SPOT, AND STEM BLIGHT OF THE EGG PLANT CAUSED BY 
 
 phomopsis vexans. In Jour. Agr. Research, v. 2, no. 5, p. 331-338, 
 1 fig., pi. 26-30. Literature cited, p. 338. 
 21) and Field, Ethel C. 
 
 1913. A DRY ROT OF SWEET POTATOES CAUSED BY DIAPORTHE BATATATIS. 
 
 U. S. Dept. Agr. Bur. Plant Indus. Bui. 281, 38 p., 4 fig., 4 pi. 
 
 22) Heald, F. D. 
 
 1896. ON THE TOXIC EFFECT OF DILUTE SOLUTIONS OF ACIDS AND SALTS UPON 
 
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 23) Hohnel, Franz von. 
 
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 PLATE 42 
 Diaporthe phaseolorum: 
 
 A. — A leaflet of Phaseolus lunatus L. , showing a number of ragged holes of various 
 sizes caused by the Lima bean podblight fungus. This specimen was collected at 
 Cape May, N. J., August 5, 1915. The disease was produced on pods by inoculation 
 with the organism (601) isolated from this leaf. Natural size. 
 
 B. — A green Lima bean pod photographed 10 days after inoculation with the ascos- 
 pore strain. Pycnidia are abundantly formed over an area about y 2 inch in diameter; 
 outside of this is another somewhat darkened area which has already been invaded 
 by the fungus, but the pycnidia have not yet broken through the epidermis. Natural 
 size. 
 
 C. — A pod showing the characteristic manner in which the fungus rapidly overruns 
 it soon after it is killed, pycnidia forming indiscriminately over the entire surface. 
 Natural size. 
 
Podblight of Lima Bean 
 
 Plate 42 
 
 \ 
 
 Journal of Agricultural Research 
 
 Vol. XI, No. 10 
 
Podblight of Lima Bean 
 
 A 
 
 Plate 43 
 
 
 Journal of Agricultural Research 
 
 Vol. XI, No. 10 
 
PLATE 43 
 Diaporthe phaseolorum: 
 
 A. — A vertical section through a pycnidium from the leaf. X 130. 
 
 B. — A vertical section through a pycnidium from a pod showing the beak and the 
 thickened dark wall surrounding it. Note also conidiophores arising from hyalin inner 
 ayer. Some spores are attached. X130. 
 
 C. — A vertical section through the epidermis and underlying host of a portion of a 
 diseased pod showing the connection and chambering of the pycnidia. X65. 
 
 D. — A somewhat diagrammatic drawing of a portion of the surface of a Lima bean 
 pod, showing the distribution of the perithecia, the shape of the beaks and their pro- 
 jection from the surface. The beaks of two perithecia may be seen just breaking 
 through the epidermis. X 16. 
 
 E. — A surface view of a portion of a bean pod showing arrangement of the pycnidia 
 and fusion of the beaks. X65. 
 
 F. — A vertical section through a perithecium from a pod. Note the dark wall 
 surrounding the perithecium in which the asci are inclosed; also the portion of the 
 beak buried below the surface of the pod. The outer end of the beak has been cut 
 away. X130. 
 
. 
 
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