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Trans. Br. mycol. Soc. 61 (2), 347-354 (1973)Printed in Great Britain

SEED-BORNE SPECIES OF MYROTHECIUMAND THEIR PATHOGENIC POTENTIAL

By THU HA NGUYEN, S. B. MATHUR AND PAUL NEERGAARD

Danish Government Institute ofSeed Pathology for DevelopingCountries, Hellerup, Copenhagen

(With Plates 36 to 38)

Myrothecium leucotrichum in cowpea, M. roridum in dahlia and nasturtium, andM, verrucaria in soybean and rice were found to be seed-borne for the first time.These are new records on their respective hosts, and their pathogenicity wasconfirmed. M. leucotrichum was the most virulent species, whereas M. roridum wasmoderate and M. oerrucaria moderate to weakly virulent. Amongst the hoststested, nasturtium was highly susceptible while pepper was immune to mostisolates of the three species. The effect of culture filtrates decreased in order ofvirulence. M. leucotrichum also produced a toxin.

On seed, sporodochia of M. leucotrichum were surrounded by long white setae,while in the other two species, sporodochia had a peripheral thick, wavy bandof white mycelium and setae were absent. M. oerrucaria was distinguished fromM. roridum by small, white contorted hyphae protruding from the sporodochia.

Species of Myrothecium, a genus belonging to the amerosporous Tuber­culariaceae, have been reported on plants of about 20 different families.The damage caused varies from host to host and from one locality toanother. There are few accounts of seed-borne infections. M. roridum Todeex Fr. was isolated by Cognee & Bird (1965) from seeds of cotton, and byRam Nath, Mathur & Neergaard (1970) from seeds of mungbean whereit caused severe leaf spots and seedling mortality. Crosier (1962) foundM. verrucaria Ditm. ex Fr. associated with seeds of barley, oat and wheat,but did not report on its pathogenicity.

There are conflicting reports on the pathogenic potentials of Myrothecium.In early papers M. roridum has generally been considered saprophytic.However, Taubenhaus (1935) isolated M. roridum from greenhouse plantsof Antirrhinum affected by a destructive crown rot which killed over 90 %of plants at Waco, Texas, in 1933 and 1934. Inoculation experiments gavepositive results. Deighton (1935) reported a Myrothecium sp. (probablyM. roridum) causing leaf spot of Impatiens holstii from Sierra Leone, Africa.Preston (1936) isolated M. roridum from pansy (Viola cornuta), and basedon 90 inoculations regarded the fungus as a potential parasite. Variationin pathogenicity among isolates of the same species was demonstrated byBrooks (1945). Fergus (1957) considered M. roridum as a saprophyte capableof invading plants that have been wounded or exposed to excessive humidityand high temperatures. Later the pathogenicity of M. roridum was emphas­ized by various workers including Barrett & Hardman (1947), Padman­abhan (1948), Munjal (1960), Littrell (1965), Ram Nath, Seth &

348 Transactions British Mycological SocietyRaychaudhury (1966), Schneider & Kuhne (1967), Schieber & Zentmeyer(1968), and Cunfer, Graham & Lukezic (1969).

M. verrucaria has been proved to be a facultative parasite on the rootsof peach seedlings at a low moisture level combined with high temperature(Wensley, 1956). It was found pathogenic to rice leaves (Singh & Aujla,1965), a new host record for India.

Comparatively very little work has been done with M. leucotrichum (Pk)Tulloch. Preston (1948) described it as M. jollymannii first from driedtobacco leaves from Nyasaland as a potential parasite since it readilyattacked leaves and stems of Viola cornuta in infection experiments.

MATERIAL AND METHODS

Isolation

Myrothecium isolates were obtained from seeds of five plant species(numbers are accession numbers of this Institute): dahlia (1280, India),cowpea (1499, India), nasturtium (1286, India), rice (1153, Egypt),soybean (1969, S. Vietnam), and another one from Egypt. Seeds wereplaced on moist blotters in plastic Petri dishes and incubated for 7 daysat 20°C under 12 h alternating cycles of near ultraviolet light and darknessand examined on the eighth day under a stereo binocular microscope(6-50 x magnification). Isolates were maintained in pure cultures onPDA. The two from dahlia and nasturtium were identified as M. roridum,the three from soybean and rice as A1. verrucaria, and the one from cowpeaas M. leucotrichum. These identifications were confirmed by the Common­wealth Mycological Institute, England. One isolate of M. oerrucariaisolated from soybean seeds, kindly supplied by Dr A. M. El Zarka, Egypt,was also used.

Morphological andcultural characters

Species were grown on PDA, and the growth rate measured after 2weeks.

Infected seeds for studies on growth characters were provided by inocu­lating nasturtium seeds with spore suspensions of the three species ofMyrothecium and incubating them at 250 for 7 days under the standardlight conditions mentioned above.

Pathogenicity tests

For testing the pathogenicity of the six isolates, five hosts (cowpea,dahlia, nasturtium, rice and sorghum) were used in preliminary leaf andsoil inoculation.

Each isolate was inoculated on the leaves of 20 young seedlings byspraying a spore suspension with an atomizer. Ten seedlings were sprayedwith sterile-distilled water as control. Spore suspensions were prepared byflooding z-week-old cultures with sterile-distilled water, scraping the fungalgrowth and straining the suspension through a clean cheese cloth to removebits of agar and mycelium. Throughout the experimental period seedlingswere maintained in a moist chamber (temperature of 24-26° and relative

Seed-borne Myrothecium. Thu Ha Nguyen and others 349

humidity of 90-95 % at night, 28-35° and 70-85 % relative humidityduring the day).

Inoculum for soil inoculation was produced on autoclaved rice grainsfor 20 days before mixing with autoclaved soil (I: 5 by volume) in pots.For each isolate 30 seeds and 30 seedlings about 20 days old grown in soilwere used, 20 healthy seeds and 20 seedlings being planted in inoculatedsoil, and 10 seeds and 10 seedlings in soil without inoculum. Pots weremaintained in a daylight room at 26-30° with relative humidity of 45-60 %.

In a second experiment cross-inoculations were made by spraying sporesuspensions of each isolate on leaves of the five original host plants and onsorghum and red pepper. The experimental procedure and conditionswere the same as in the preliminary experiment. Besides the above numberof seeds and seedlings used in the first test, 30 more seedlings developedon blotters were used for each isolate, including those kept as control.

For both experiments, the final observations on leaf and soil inoculationswere recorded after 2 weeks and 3-4 weeks respectively.

Filtrate tests

These tests were carried out in order to study the nature ofwater-soakedlesions observed in the first experiment on the leaves of inoculated plantsof cowpea, nasturtium and dahlia, and to confirm and compare the toxiceffect of filtrates of the three species.

Filtrates were prepared from M. leucotrichum (cowpea), M. roridum(nasturtium), and M. verrucaria (soybean from S. Vietnam). Cowpea,nasturtium and soybean were used as test plants. The basic medium wasDifco Czapek Dox broth, pH 7'3, after sterilization. Cultures were incu­bated at 25° and relative humidity of 45-50 %. The three species weregrown for 20 days in standing cultures in Erlenmeyer flasks, each containing100 ml of the basic medium. The mycelial mats were removed by filtration,the pH adjusted to 7, and the following dilutions made with sterile­distilled water: I: 2, I: 5, I: ro, I: 50, I: roo. The effect of the variousdilutions of culture filtrates was studied on cut shoots, approximately25 em high.

RESULTS

Growth onseeds andin culture

Myrothecium leucotrichum. Sporodochia on the seed are circular, discoid,often coalescing to form continuous black masses, individual sporodochiasurrounded by a white fringe of long straight setae (PI. 36, fig. I). OnPDA fructifications develop in concentric rings, initially olive in colour,later turning jet black. Diameter of a z-week-old colony 5'3 ern (PI. 37,fig. 3). Conidia one-celled, cylindrical with rounded ends, hyaline to lightgreen, 6-II x 1'4-2'5 flm.

M. roridum. Sporodochia on the seed are often confluent, viscid, globularor conical, flattened or slightly concave, with a white margin of flocculentcontorted hyphae, setae lacking (PI. 36, fig. 2). On PDA sporodochia areproduced in abundance, usually on the substratum, less among the looseaerial hyphae. Diameter of a z-week-old colony 5'8 ern. Fructifications

M YC 61

35° Transactions British Mycological Societydevelop in concentric rings, green at early stage of growth (PI. 37, figs.I, 2). Conidia one-celled, cylindrical with rounded ends, hyaline to lightgreen, becoming black in mass, 5-7 x 1'2-2 flm.

M. verrucaria. Sporodochia on the seed are surrounded by a white rimof flocculent contorted hyphae, setae lacking. The species can be differ­entiated from M. roridum particularly by the presence of short, contorted,white hyphae emerging from the viscid spore mass (PI. 36, fig. 3). OnPDA sporodochia are predominantly in the loose white mycelium, lesson the substratum. The diameter of the colony (PI. 37, figs. 4-6) after2 weeks 6'5 em, Conidia one-celled, ovoid or elliptic, subhyaline to paleolive, 5-7 x 2'5-3'3 /lm.

Pathogenicity tests

Symptoms. In leaf inoculations all three species of Myrothecium producedsimilar symptoms on the different hosts. Numerous minute, circular,purplish brown to black leaf spots were a common feature, particularly innasturtium (PI. 38, fig. 3). General browning of the veins was produced.On roots and stem bases the three pathogens produced similar symptoms.Severely affected plants developed necrotic lesions on the stem bases andextended rot in roots; the infected plants wilted and eventually died. Lessseverely attacked plants became highly stunted, the roots only partlyrotted and developed mild browning, and the plants were not killed.

Effect of culture filtrates. In symptoms produced by culture filtrates ofthe three pathogens, veinal necrosis was particularly pronounced (PI. 38,fig. 5). The dilutions I: 2, I: 5, I: 10 of culture filtrates of the three speciesseverely affected soybean and nasturtium, whereas dilution of I: 50 hada moderate, and that of I : 100 a very mild effect. In cowpea the dilutionsof I: 2, I: 5, I: 10 induced mild symptoms, whereas I: 50 and I: 100

dilutions produced no symptoms at all. Cowpea leaves showed discolouredveinal necrosis near the base, and wilted. On nasturtium the veins andleaf tissues along the veins became discoloured, turned yellow-green, andlater the leaves developed a water-soaked appearance. Discoloration wasmore pronounced in the interveinal areas than in the veins. On soybeanthe leaves had pronounced veinal necrosis. They finally dried but thecolour was not much affected.

There were definite differences in degrees of effect exercised by thethree filtrates. The filtrate from M. leucotrichum had the strongest effectproducing severe veinal necroses on soybean and cowpea, while thefiltrate from M. roridum produced severe veinal necrosis on the main veinsonly, the smaller veins being less affected. Finally, the symptoms producedby the filtrate from M. verrucaria were faint in both hosts, on cowpea veinalnecroses being developed only at the bases of leaf lamina. The severity ofthe symptoms produced by the filtrates on nasturtium was almost equalfor all the three species of Myrothecium (PI. 38, fig. 5).

Virulence of the pathogens. The degree of pathogenicity of the isolatesstudied, and the susceptibility of the hosts were defined by grading theinfections (Tables I and 2). The virulence of the isolates may thus bepresented as follows: highly pathogenic - M. leucotrichum (cowpea) andM. roridum (nasturtium); moderately pathogenic - M. verrucaria (soybean,

Seed-borne Myrothecium. Thu Ha Nguyen and others 351

T able 1. Rating ofinjection of Myrothe cium spp. using the leafinoculation method

Red Soy- Nas- Viru-Isola tes (host) pepp er Dahlia bean R ice Sorghum tur tium Cowpea len ce

Mi leucotrichum 0 3 2 2 3 3 3 16(cowpea)

11,'1. roridum (da hlia) 0 3 2 2 3 12AI. roridum 0 3 3 2 3 3 15

(nasturtium)M. oertucaria (rice) 0 2 3 2 10

M . t errucaria (soy- 0 2 2 3 I 10bean - Egypt )

M . uertucaria (soy- 0 2 3 2 2 3 2 14-bean - S. Vietnam)

Susceptibility 0 10 13 12 II 17 14

Table 2. Infection ofMyrothecium spp. on the original host plantsusing the soil inoculation technique

Isola tes

AI . leucotrichumM . roridumM . roridumM, oerrucaria (Egyptian isolate)M. oerrucaria (S. Vietna mese isola te)M, t'trrucaria

I-lost

CowpeaDahl iaNasturtiumSoybeanSoybeanR i<.e

Rating

333I

I

3

No infection , 0 ; mild infection, I ; mod erat e infection, 2 j and severe infection , 3.

S. Vietnamese isolat e) and M. roridum (dahlia) ; weakly pathogenic­M . oerrucaria (rice and soybean, Egyptian isolate).

In the soil inoculation test there was little difference in pathogenicitybetween the isolates , although M. oerrucaria from soybean induced mildsymptoms only.

In decreasing ord er of susceptibility the test plants may be arranged ashighly susceptible (nasturtium and cowpea); moderately susceptible (soy­bean, rice, sorghum and dahlia), and immune (red pepper).

DISCUSSION

Seed transmission of Myrotheciumspecies has not been adequately investi­gated. This may be in part due to the low percentages of infection encount­ered in seeds and by the conflicting reports regarding pathogenicity. RamNath et al. (I970) observed M. roridum on I-3 % of seeds of Phaseolusaureus from India. When introduced into the soil th e fungus attacked plantsseverely, and spore suspensions caused severe leaf spots in young seedlings .

The three species considered in the present investigation have also beenisolated from samples with very low incidence of infections; M. roridumfrom two out of 400 seeds of dahlia, M. uetrucaria from one seed out of 4 00seeds each of rice and soybean; and M. leucotrichum from one seed out of400 seeds ofcowpea . It is the first time that M. leucotrichum and M, oerrucatiahave been recorded as seed-borne. The isolates were classified as highlyviru lent, moderately virulent, and weakly virulent.

352 Transactions British Mycological SocietyIn leaf inoculations soybean was severely infected by M. verrucaria from

the same host but only mildly in soil inoculations. The actual amount ofinoculum involved in causing symptoms is probably important. Leaves ofsoybean are hairy and retain large numbers of spores, in contrast to theother test plants. Soybean roots are tough, those of cowpea and nasturtiumtender. These facts may explain why leaves of soybean are severely attackedwhile the roots remain unaffected but the suggested explanations foraetiology need further investigation.

On the other hand, the rice seedlings were only moderately damagedafter leaf inoculations, but severely attacked in infested soil. Also here theactual amount of inoculum must be considered. Rice leaves stand moreor less vertically, retaining little of the spore suspension used as inoculum,whereas the roots are thin, long and easily injured during transplanting,and consequently more subject to severe infection. Seedlings from seedspreviously germinated on blotters and transplanted into the infested soilas well as those from direct sowing in infested soil became stunted. Thosegrown first in soil and then transplanted to inoculated soil were killed.

M. leucotrichum proved to be the most potent pathogen, whereas M.verrucaria was the weakest. This is in agreement with the observations ofPreston (1936) and Cognee & Bird (1965). Recently it was shown byBraverman (1971) that leaf spot of bird's-foot trefoil (Lotus corniculatus)caused by M. verrucaria is prevalent in the United States. Greenhouse testsindicated that cultivars of L. corniculatus were slightly to moderately sus­ceptible, while L. major and L. pusillus were moderately resistant, and L.arabicus and L. maroccanus highly susceptible.

Red pepper was immune to all three species of Myrothecium and also theculture filtrates from these species did not cause any damage in red pepper.On the other hand, they induced vein-clearing in dahlia, veinal necrosisin soybean, and water-soaked lesions in nasturtium.

The results from the filtrate tests were in agreement with those fromthe infection experiments. The effect of the filtrates of the three speciesdecreased in the same order as the virulence - M. leucotrichum, M. roridum,M. verrucaria.

This is the first record in the literature of toxin production by M. leuco­trichum, although the two other species are known to produce phytotoxins.Vittimberga (1963) reported Muconomycin A from broth cultures of M.verrucaria, and Harri et al. (1962) established Roridin A and B as toxinsproduced by M. roridum. Toxicity of Necrocitin, an antifungal antibioticand plant toxin isolated from M. roridum, was demonstrated experimentallyby Pawar, Deshmukh & Thirumalachar (1965).

REFERENCES

BARRETT,]. T. & HARDMAN, D. A. (1947). Myrothecium leafspot and canker of gardenia.Phytopathology 37, 360.

BRAVERMAN, S. W. (1971). Myrothecium leafspot of birdsfoot trefoil. Phytopathology 61, 127.BROOKS, F. T. (1945). Notes of the pathogenicity of Myrothecium roridum Tode ex Fr.

Transactions oftheBritish Mycological Society 27, 155~157.

COGNEE, M. & BIRD, L. T. (1965). La pathogenic de Myrothecium roridum Tode sur Iecotonnier. Coton etfibres tropicales 20, 343-350.

Seed-borneMyrothecium. Thu Ha Nguyen and others 353CROSIER, w. F. (1962). Glomerella angulata and Myrothecium spp. in seeds of small grains.

Plant Disease Reporter 46, I I I-I 13.CUNFER, B. M., GRAHAM, J. H. & LUKEZIC, F. L. (1969). Studies on the biology of

Myrothecium roridum and M. verrucaria pathogenic on red clover. Phytopathology 59,1306-1309.

DEIGHTON, F. C. (1935). Mycological work. Report of the Department of Agriculture, SierraLeone 1935, 22-26.

FERGUS, C. L. (1957). Myrothecium roridum on gardenia. Mycologia 49, 124-127.HARRI,E., LOEFFLER, W., SIGG, H. P., STAHELIN, H., STOLL, C., TAMM, C. & WIESINGER,

D. (1962). Uber die Verrucarine und Roridin, eine Gruppe von cytostatischhochwirksamen Antibiotica aus Alyrothecium-Arten. Helvetica chimica Acta 45,839-853.

LITTRELL, R. H. (1965). A Myrothecium rot of gloxinias. Plant Disease Reporter 49,78-80.

MUNJAL, R. L. (1960). A commonly occurring leaf spot disease caused by Myrotheciumroridum Tode ex Fr. Indian Phytopathology 13, 150-155.

PADMANABHAN, S. Y. (1948). Occurrence of Myrothecium roridum Tode ex Fries on cowpeain India. Current Science 17, 56-57.

PAWAR, V. H., DESHMUKH, P. V. & THIRUMALACHAR, M.J. (1965). Necrocitin: a newcrystalline antifungal antibiotic and plant toxin. Hindustan Antibiotics Bulletin 8,60-63.

PRESTON, N. C. (1936). The parasitism of Myrothecium roridum Tode. Transactions of theBritish Mycological Society 20, 242-250.

PRESTON, N. C. (1948). 0 bservations on the genus Myrothecium. II. Myrothecium gramineumLib. and two other species. Transactions of the British Mycological Society 31,271-276.

RAM NATH, MATHUR, S. B. & NEERGAARD, P. (1970). Seed-borne fungi of mungbean(Phaseolus aureus Roxb.) from India and their significance. Proceedings of the Inter­national SeedTesting Association 35,225-241.

RAMNATH, SETH, M. L. & RAYCHAUDHURY, S. P. (1966). Anew blight diseaseofbrinjalcaused by Myrothecium roridum Tode ex Fr. Indian Phytopathology 19, 224-225.

SCHIEBER, E. & ZENTMEYER, G. A. (1968). Myrothecium stem necrosis and leaf spot:Important coffee disease in Guatemala. Plant Disease Reporter 52, I 15-I 17.

SCHNEIDER, R. & KUHNE, H. (1967). Myrotheciumroridum als Erreger Trieb- und Stengel­faule an Hypocyrta glabra in Deutschland. Nachrichtenblatt des Deutschen Pflanzen­schutzdienstes, Stuttgart 19, 75-77.

SINGH, M. & AUJLA, S. S. (1965). Myrothecium verruearia Ditm. ex Fr. on rice in Punjab.Journal ofResearch. Punjab Agricultural University Ludhiana 2, 120-12 I.

TAUBENHAUS, J. J. (1935). On a black crown rot of greenhouse snapdragons caused byMyrothecium roridum Tode. Phytopathology 25, 969-970.

VITTIMBERGA, B. M. (1963). The Muconomycins. I. Studies on the structure of Mucono­mycin A, a new biologically active compound. Journal of Organic Chemistry 28, 1786­1789.

WENSLEY, R. N. (1956). The peach replant problem in Ontario. IV. Fungi associatedwith replant failure and their importance in fumigated and non-fumigated soils.Canadian Journal ofBotany34, 967-98 I.

EXPLANATION OF PLATES 36 TO 38PLATE 36

Growth of the three species of Myrothecium on seeds of nasturtium.Fig. I. M. leucotrichum, Sporodochia surrounded by a rim of white long straight setae (x go).Fig. 2. M. roridum. Sporodochia often confluent, surrounded by a thick band of white mycelium(x go).Fig. 3. M. oerrucaria. Growth characters similar to M. roridumwith the exception ofshort contortedhyphae among the mass ofspores seen particularly well in (b).(a, x go), (b, x 580).

354 Transactions British Mycological Society

PLATE 37Two-week-old cultures of Myrothecium species on potato dextrose agar.

Figs. 1,2. AI. roridumFig. 3. M. leucotrichum (syn. M.jollymannii).

Figs. 4, 5, 6. M. verrucaria.

PLATE 38Leaves infected by Myrothecium species and showing symptoms.

Fig. 1. Cowpea leaf inoculated with M. leucotrichum. Symptoms 5 days after inoculation.

Fig. 2. Symptoms of M. roridum (dahlia isolate) on leaf of dahlia 8 days after inoculation.

Fig. 3. Symptoms of M. roridum (nasturtium isolate) on nasturtium leaf 8 days after inoculation.

Fig. 4. Mild symptoms (veinal necrosis) in leaf of soybean inoculated with M. verrucaria 7 daysafter inoculation.Fig. 5. Typical symptoms produced by the culture filtrates of Myrothecium on nasturtium leaf.The symptoms produced by any of the three species studied were alike.

(Acceptedfor publication 2 March 1973)

T rans. Br. mycol. Soc. Vol. 6 1. Plate 36

(Facing p. 354)

Trans. TIr. mycol. Soc. Vol. 61. Plate 37

Trans. Br. mycol. Soc. Vol. 51. Plate 38

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