The perfect state of Nigrospora oryzae

[ 355 ] Trans. Brit. mycol. Soc. 46 (3), 355-360 (1963).

THE PERFECT STATE OF NIGROSPORA ORYZAE By H.]. HUDSON

Botany School, University of Cambridge (With

I

Text-figure)

The perfect state of Nigrospora oryzae (B. & Br.) Petch is described as a new genus of the Sphaeriales. Dimensions of conidia in cultures from single ascospore isolates, conidial isolates and cultures of N. oryzae, N. sphaerica and N. sacchari are compared.

Mason (1927, 1933) has discussed the genus Nigrospora Zimm. and pointed out that the numerous nominal species apparently differ only in spore size. He listed three species: N. oryzae (B. & Br.) Petch, with aleuriospores r o-rfiu, average near 14ft; N. sphaerica (Sacc.) Mason, with aleuriospores 14-20fL, average near 16fL; and N. sacchari (Speg.) Mason, with aleuriospores 15~25 fl. The conidial dimensions given in the type diagnoses for these three species are 12'5-15, 16-18 and 20-22 x 15-18 fL respectively. Mason (1933) measured 50 conidia from type material of each species. His measurements are 11-16, 15-19 and 16-24 fL, respectively. There is evidence that N. oryzae and N. sphaerica can be mistaken for each other in culture. Mason quoted a culture of N. sphaerica which started with spores 15-20 fL diam. and in the first subculture no spores greater than 16 fL diam. were abserved. Apart from these three species and N. panici Zimm., the type species, McLennan & Hoette (1933) described two new species, N. musae, with aleuriospores 18 x 15 fL, as the causal agent of' squirter' disease of bananas, and N. canescens, a non-pathogenic form with aleuriospores 19 x 17 fl. Both occurred on flecked and speckled banana leaves. The authors compared N. musae and N. sphaerica in culture and the two species appeared distinct. They also tested the pathogenicity of N. musae to banana fruit and could produce 'squirter' symptoms in the laboratory artificially. Bananas inoculated with N. sphaerica developed a soft brown rot resembling that induced by N. musae but more restricted in extent. Simmonds (1933) referred the causal agent of' squirter' disease to N. sphaerica. It is evident from his findings that N. sphaerica consists of a number of different strains which merge with N. sacchari. The conclusions of Simmonds are considered in detail by Wardlaw (1935, 196I), whose findings could explain the fact that McLennan & Hoette found marked cultural differences between N. musae and N. sphaerica in culture and on bananas. Prasad, Agnihotri & Agarwal (1960) described N. padwickii on Oryza sativa with much larger spores (37'94 x 34.82 fL) than any other species. ]echova (1963) has placed all species with conidia within the range I 1-19 x 9-18ft in N. maydis (Garov.) ] echova, This would include all the species mentioned above except N. panici and N. padwickii. She has accepted 23-2

356

Transactions British Mycological Society

as a basionym Sporotrichum maydis Garov. The type diagnosis for this species gives conidial dim ensions as I4-IS j.£. Mason (1927) stated that there is no type material in existence of S. maydis and it would seem better to accept his interpretation of the species. M eredith (1961) found that Nigrospora spores were regular components of the air spora of J am aican banan a plantations and concluded that the dominant species trapped were N. oryzae and N . sphaerica, whereas N . sacchari was infrequ ent, because spores measuring I3-1 8 j.£ predominated on his spore traces. Hudson (1962) described the distribution of N. sphaerica on ageing leaves of sugar can e. This specific epithet was used although the range of spore size found would include several described species. Most single conidial isolat es behaved in cultures as N. sphaerica (Table 2). Du ring the course of this study a Pyrenomycete was collected on Saccharum oificinarum, Vetiveria zizanioides, Panicum maximum and M usa sp. Single ascospores from each of these collections produced Nigrospora conidia in culture (T able 1). Conidia developing naturally on the hosts (Table 1) ranged from 12 to 21·Sj.£ and from ascospore cultures 12-19j.£, which would suggest, on preliminary examination, identification as N. sphaerica with overlap to N. oryzae on th e one hand and N. sacchari on the other. Table

1.

Dimensions (j.£) of ascospores and conidia Conidia from cultures

H er b, IMI no ,

Ascospor es on the host

Vetiveria zizanioides

79239 (1)* (2) (3) (4)

16-18'5 x 4'5 -5" 5 16-18'5 x 4' 5-5"5 16-19'5 x 4'5-6 17- 19' 5 x 4'5-6 16-2 1 x 5-7

Saccharum officinarum

79 237 (I)

18--19'5 x 5"5-6 '5 16-2 1'5 x 5- 7

Host

Con idia on th e host

of

of

ascospores

coni dia

13- 16 '5 12-15 13-16 13- 15 13- 17 12- 21 (13-16)

Panicum maximum

79238 ( I) (2)

79 24° (I)

14- 17

14- 17'5 12- 19 13- 17 15- 17

14- 19' 5 15- 2 1 13- 17 14- 17 13- 17'5

17- 19'5 x 4'5- 5"5 16-1 8 x 4"5-6 16-1 9 x 4'5-6

13- 20

13- 21'5 (16- 19) 14- 18

Musa sapientum

Conidia from cultures

13- 16 14- 16

16-22'5 x 5-6'5 17- 20'5 x 4'5-6 14-21' 5 (16-18)

* Numbers in parentheses repr esent different perith ecia from

the same collection,

The following descrip tion of the perfect state is based on a collection on Vetiveria zizanioides (Herb. IMI 79239). Perithecia are formed in clusters of 1-7 in uniseriate rows or ar e irregularly arranged and up to 2 mm. long. They develop beneath th e epidermis but become erumpent eventually with papillate ostioles, most of the

Nigrospora oryzae. H. J. Hudson

357

perithecia projecting above the leafsurface (Fig. 1A) . There is a blackened area of host tissue round each group. The perithecia are globose or ovoid

and up to

250~ diam. The ,hort:Y . ": ',~",

'eedGa~ni()te~te,

: ;~.: : :..: .. .

A

200p

B

20p

c)DDDG DDDDO

He!

Fig. I. Khuskia oryzae. A, Pcrithecia ; B, asci and paraphyses; C, ascospores, all from Vetiveria zizanioides; D, ascospores from Saccharum ojJicinarum ; E, discharged ascospores ; F, germinating ascospores pr oducing Nigrospora conidia ; G, conidia from V. zizanioides; H , conidia from an ascospore culture from S. ojJicinarum.

8-spor ed, 55-75 X 8'5-12 iL, and surrounded by numerous, thin-walled, septate paraphyses. They are up to 3 iL wide and longer than the asci (Fig. 1B). The hyaline, granular ascospores ar e curved, inequilateral, taper to the base and have rounded ends. They are initially unicellular but after

358

Transactions British Mycological Society

discharge from the ascus and on germination usually develop a single transverse septum which divides each ascospore unequally into two cells (Fig. I E). They are arranged irregularly biseriately in the ascus and measure 16-2 I x 5-7 p,. On 2 % malt agar germ tubes are formed after 36 hr. and grow rapidly, producing a submerged growth of radiating hyphae up to 9 p, diam. From these hyphae a woolly aerial mycelium is formed. On oatmeal agar this becomes rapidly pigmented near the surface ofthe medium and within giving a black coloration to the culture. Jet black, shiny aleuriospores, globose in end view and ellipsoidal in side view and measuring 13-17 P, are formed scattered over the surface or in clusters. These are typical of the form-genus Nigrospora (Fig. I H). On oatmeal agar large black sclerotial bodies develop which later produce a number of protruding necks resembling perithecia, but no asci have ever been found in these. When ascospores are massed together in culture, such as when they are shot off directly from perithecia on to an agar plate, many produce Nigrospora conidia directly (Fig. I F). This perfect state is described as a new genus as it does not match any known fungus genus, though it bears resemblances to several members of the Polystigmataceae of the Sphaeriales. Khuskia gen.nov. Perithecia aggregata, atra, globosa vel ovoidea, ostiolo papillato, erumpente. Asci clavati, stipite brevi, unitunicati, octospori. Paraphyses septatae, muros tenues habentes, longiores atque asci. Ascosporae hyalinae, granulosae, curvatae, inequilaterales, ad finem inferiorem tenues, irregulariter biseriatae, continuae, germantes uniseptatis simul inaequabiles duas cellas habentes. Species typicus Khuskia oryzae Hudson est.

Khuskia oryzae sp.nov. (Fig. I) Perithecia 1-7 in soros aggregata, atra, globosa vel ovoidea, ostiolo papillato, ad 250 jJ. diam. Asci clavati, stipite brevi, octospori, 55-75 jJ. longi et 8'5-12 jJ. lati. Paraphyses septate, ad 3 jJ. latae, muros tenues habentes, longiores atque asci. Ascosporae hyalinae, granulosae, curvatae, inequilaterales, ad finem inferiorem tenues, irregulariter biseriatae, continuae, gennantes uniseptatis simul inaequabiles duas cellas, 16-22 jJ. longae et 5-7 jJ. latae. Status conidiatis Nigrospora oryzae est. Habitat. In culmis et foliis emortuis Vetiveriae zizanioides in campo Collegiae Universitatis, Indies Occidentis, Jamaica. Typus: Herb. IMI 79239.

Since a number of species of Nigrospora have been erected apparently solely on the character of spore dimensions, and the range of spore size for the whole complex is a very limited one (ro-25JL, excluding N. padwickii), it was felt desirable to study a number of isolates of the various species in culture on different media to assess the range of conidial dimensions within each species and to compare this range between species. For this purpose single ascospore isolates from Vetiveria ;;.i;;.anioides (IMI 79239) and from Panicum maximum (IMI79238), and two isolates from single Nigrospora spores from sugar cane were compared with cultures of N. sacchari (IMI77472), N. oryzae (IMI82764) and two isolates of N. sphaerica (IM146685 and one from C.B.S.). It was unfortunate that a culture of N. musae (C.B.S.) and of N. panici (IMI 78285) produced only

Table

2.

Conidial dimensions and means of various Nigrospora isolates on different media and substrata (Measuremen ts and means of fifty conidia .)

Herb, IMI no, 79239, Khuskia otyz ae 79238, K, oryz ae Nigrospora, no , I from suga r cane Nigrospora, no , 2 from sugar cane 77472, N , sacchari 82764, N , oryzae 46685, No sphaerica

C,B ,S., No sphaerica

Steri le sugar cane leaves

Sterile oat stems

Oatmeal agar

2% ma lt

12,8-16'4 (14' 7) 14,6- 18'2 (15' 2) 16'4- 20'0 (17'8 ) 16'4- 20 '0 (18'7) 16'4-21,8 (18'5) 14,6-18'2 ( 16'7) 14'6-20'0 (17,6) 14'6-20'0 (In)

12'8-1 8'2 (15'3) 1.1-'6-18'2 (16 '2) 16'4- 20' 0 (18'5) 14'6-20'0 (17'5) 16'4-21,8 (19'2) 12'8-18'2 ( 15'0) 16' 4- 20' 0 (18'2) 14,6-2 1,8 (19 '2)

14'6-20'0 ( 15,6) 12'8-18'2 ( 15'0 ) 16 '4- 20'0 ( 18'3 ) 16'4- 20 '0 (18' 2) 16' 4- 2 1,8 (18 '3) IO'g-18'2 (13'7) 16 '4- 20 '0 (18'7) 14'6-20'0 ( 17'0)

Z

agar

Potato dex tros e agar

G lucose phosphate agar

Over-a ll

12'8-18'2 (15 '1) 14,6-18'2 (16' 7) 16'4-20'0 (18' 1) 14,6-21,8 ( 18'3) 18'2-21'8 (18'5 ) l o '9-1 4 '6 ( 13'3) 16'4-20'0 (17'3) 16'4- 21'8 (18'1)

12'8- 16'4 (14,6) 14,6-18'2 ( 16 '0 ) 16' 4- 20 '0 ( 18'2 ) 16'4-2 1'8 (18'3) 16'4-21'8 ( 18'1) 12,8-1 8'2 ( 14'3) 16 '4- 21'8 (18 ,6) 16' 4- 21'8 (18, , )

12'8-16'4 ( 15 '3) 14,6-20'0 (16 '2) 16'4- 2 1'8 (17'8) 14'6-20'0 (17' 1) 18'2-23'8 (20'7) 12'8-1 8 '2 ( 16'2) 16 '4- 20 '0 (17,6) 14'6-20'0 (17'4)

12'8-20'0 (15'1) 12'8-20' 0 (15'9) 16'4 - 2 1'8 (18'1 ) 14'6-21,8 (18 '0 ) 16'4- 23'8 (18'g) 1O'g-18'2 (14 '9 ) 14'6-2 1'8 (18'0) 14'6-21,8 (17'9)

~,

8 C/.l

"0 0

'"1

P:l

0

~P:l ~

;t: ~

~

t

UJ Vl

\0

360

Transactions British Mycological Society

very sparse conidia and could not be used. A selection of the results obtained is given in Table 2. The two isolates from Nigrospora conidia from sugar cane are closely similar to the two cultures of N. sphaerica in spore dimension range and means on the various media and the N. sacchari culture shows considerable overlap with these. The two single ascospore cultures compare closely with the N. oryzae culture and have considerably smaller spores. Though there is overlap in conidial dimensions, the means are different. For this reason N. ory;::ae is considered distinct, in accord with Mason (1933); the spore dimensions I obtained of both N. oryzae and N. sphaerica, however, are slightly larger than those quoted by Mason. This conclusion is also in agreement with Webster (1952) who gives spore measurements of 18-21 x 14-15 fL for N. sphaerica. The N. sphaerica used from C.M.I. was Webster's isolate. During the course of this study and the previous one many Nigrospora isolates were handled. All fell into either the larger- or the smaller-spored group. Each of these groups was sufficiently distinct and stable enough to be ascribed to two species, the larger-spored N. sphaerica and the smallerspored N. ory;::ae. I am indebted to Dr C. Booth for his assistance and encouragement. I am grateful to the Curator of Fungus Cultures at the Commonwealth Mycological Institute for maintaining cultures of Nigrospora for me and supplying others. REFERENCES

HUDSON, H. J. (1962). Succession of micro-fungi on ageing leaves of Saccharum ojjicinarum. Trans. Brit. mycol. Soc. 45, 395-423. JECHOVA, V. (1963). Nove druhy rodu Nigrospora ajimi ptrsobene kniloby na dovazenern jiznim ovoci. Ces. Mykol. I7, 12-20. MCLENNAN, E. I. & HOETTE, S. (1933). Nigrospora musae n.sp. and its connection with 'squirter' disease of bananas. Bull. Coun. sci. Res. Austr. no. 75, 36 pp. MASON, E. W. (1927). On species of the genus Nigrospora Zimmerman recorded on monocotyledons. Trans. Brit. mycol. Soc. I2, 152-165. MASON, E. W. (1933). Annotated account offungi received at the Imperial Mycological Institute. List II, Fasc. 2. Mycol. Pap. no. 3, 67 pp. MEREDITH, D. S. (1961). Atmospheric content of Nigrospora spores in Jamaican banana plantations. ]. gen. Microbiol. 26, 343-349. PRASAD, N., AGNIHOTRI, J. P. & AGARWAL, J. P. (1960). A new species of Nigrospora Zimm. on Oryza sativa. Curr. Sci. 29, 352-353. SIMMONDS, J. H. (1933). Squirter disease of bananas. Qd agric. ]., 39, 8-10. WARDLAW, C. W. (1935). Diseases of the banana. London: Macmillan & Co. Ltd. WARDLAW, C. W. (1961). Banana diseases. London: Longmans, Green & Co. Ltd. WEBSTER, J. (1952). Spore projection in the hyphomycete Nigrospora sphaerica. New Phytol. 5I, 229-235.

(Accepted for publication 16 September 1962)