TAXONOMY AND DISCUSSION ON THE NOMENCLATURE OF MACROPHOMINA PHASEOLI (MAUBL.) ASHBY, AND ITS ISOLATES FROM INDIA

by

N. B. KULKARNI & B . C. PATIL 1

Plant Pathological Laboratory, College o[ Agriculture, Poona-5

(18.I.I965)

ASHBY (1927) proposed the binomial of the fungus Rhizoctonia bataticola (TAuB.) BUTLER inciting charcoal rots, blights, stem rots and root-decays as Macrophomina ])haseoli (MAuBL.) ASHBY mainly on the basis of the characters viz. (i) pycnidial development devoid of stroma, (ii) size of pycnidia, ostiole, pycnidiospores (iii) ratio of L : B of the spores nearing 3 : 1 and (iv) pycnidiospores long, narrow, thin walled, elliptical, remaining hyaline and continuous. He considered Macrophoma phaseoli MAUBL. (1905), Sclerotium bataticola TAUB. (1913), Macrophoma corchori SAW. (1916), Macro- phoma cajani SYd. & BUTL. (1916), Macrophomina phil@pinensis PETR. (1923), Rhizoc~onia lamelli/era S~ALL (1924), Rhizoctonia bataticola (TAuB.) BUTL. (1925), Dothiorella ca]a~,i SYD. & BUTL. (1925), ~//acrophoma sesami SAW. (1922), as synonyms. The genus Macrophoma, BERL. & VOGL. and the species M. phaseoli reported by )/[AUBLANC (t905) under it, were rejected by ASHBY (1927} for pycnidial forms of R. bataticola mainly on the grounds that this genus Macr@homa was considered to be a heterogeneous group, as most of the species of Macrophoma were considered by PETRAK & SYDOW (1926) as belonging to other genera, viz. Dothiorella SACC. and Botryodiplodia SACC. in particular, reducing it into a monotypic genus with a species Macr@homa pinea (DEsk.) PeT. & SYD. retained under it. This species develops pycnidia on stroma and has larger spores 3 0 - - 5 0 # × I I - -20#. The objections raised for the genus Macrophoma also hold good for the genus Dothiorella. GOt- ~)ANICH (1947) confirmed the authenticity of the genus Macro- phomina in comparison to Dothiorella. The genus Macr@homi~,a differed from Dothiorella in the absence of a typical extra-matrical

1) Present address of the senior author is: Wheat Rust Mycologist, Wheat Rust Res. Station, Mahabaleshwar (India).

258 N . B . K U L K A R N I ~ B . C. P A T I L

stroma in the pycnidia. The binomial Macr@homina l~haseoli (MAoBL.) ASHBY as proposed by ASHBY (1927) for the earliest pycnidial form on beans after MAUBLANC (1905), was changed to M. l~haseolina (TAssI) G. GOIDANICH, by GOIDANIC~I (1947), giving priority to the original matrix collected by TAssI in 1901 and examined later b y him. Other synonyms of this new binomial were 3iacrophoma phaseoli MAUBLANC, Dothiorelta cafani SYD. & BUTLE.% D. ~hilippinensis PETR. and Macrophomina phaseoli (MAuBL.) ASHBY. This binomial proposed by GOIDANICH (1947) appears to be incorrect since ASHBY (1927) should be given the priority for creating a variety as his observations were made earlier to those made by GOIDA•ICH. Thus, it is felt that the validity of M. phaseoli (MA~JBL.) ASHBY stands. The genus Botryodiplodia which is considered to be very close to Macrophomina can be distin- guished on the basis of immature, hyaline, thick walled or mature bicellular, thick walled and coloured pycnidiospores having L : B ratio of 2 : 1 or lower as pointed out by SHAW (1924) and EDWARD (1954). THIRUMALACHAR (1953) considered the isolates of M. phaseoli (MAu~L.) ASHBY from jute, potato, maize stalks, garlic and castor belonging to Botryodiptodia since the pycnidiospores when kept for germination become thickened and cotoured with the development of septa. EDWARD (1954) did not agree with this change when he compared typical culture of Botryodiplodia and culture of M. phaseoli from potato and showed that Botryodiplodia isolates caused infection and incited pycnidia on potato stems. Tile pycnidiospores were decidedly thick walled, bicellular with a ratio of 2 : I for L : B which fact was not so for M. ~bhaseoli isolate. On basis of his findings he considered that Botryodiplodia and Macrophomina are two distinct genera and transfer of M. phaseoli species to Botryodiplodia is unwarranted.

As reported by REICHERT • HELLI~ICER (1947), SAREJANNI & CORTZAS (1935), YOUNG (1949), UI'PAL (1935), LIKmTE (I936), THIRUMALACHAR (1953) and several other workers, M. phaseoli incites disease on a variety of host plants, widely distributed in different agro-climatic zones of several countries and this is consider- ed as a cosmopolitan fungus. It is expected that a fungus having such a wide distribution and host range, should exhibit some differ- ences in its morphology, and growth behaviour, when studied in different countries and also from different unrelated hosts. If this presumption is correct, then the findings of REICHERT & HEL- LINGER (1947), HAIGI-I (1930) and other workers on M. ~haseoli require re-examination in the light of the studies undertaken on the four isolates of M. ~haseoli obtained from cotton, castor, groundnut (Arachis hypogaea L.) and sesame.

These isolates when tested for pathogenicity and cross-inoculation studies on 19 hosts did not show- any differences, confirming the findings of BUTLER (1918) for cotton and groundnut isolates, of PICASAD (1944) for cotton, castor and sesame isolates and of COOK

N O 3 I E N C L A T U R E O F M A C R O P H O M I N A P I ' I & S E O L I 259

(1945) for castor isolate of M. phaseoli. These findings, however, are not in agreement with those reported by SUNDARARAMAN (1932) for sesame and groundnut isolates, by REICttERT & HELLINGER (1947) for sesame isolate and also for cotton isolate (Anonymous 1942). As has already been reported by several authors, pathogenicity of M. phaseoli is dependent on the stage of the fungus used at the time of inoculation and also the technique. The findings of THIRU- MALACtIAR (1953), MONIZ (1961) and also the results obtained by the present authors, clearly show that the stage of the fungus growth i.e. young and actively growing mycelium, is very important in inciting the infection by this fungus. The other considerations of equally important nature are the suitable technique for creating infection and favourable temperature and humidity requirements. By following the techniques, developed now, under 28°--30°C temperatnre conditions and adequate watering of the pots, no difficulty was experienced in getting the infection by all the isolates of M. phaseoli used in the present studies. Although reports by several workers, including that of MoNIz (1961) mentioning therein non-pathogenic behaviour of some M. phaseoli isolates, have been made, it is likely that for want of suitable technique, the stage of fungus growth at the time of inoculation and environmental condi- tions, pathogenicity was not obtained and this might have led these authors to consider the non-pathogenic behaviour of M. phaseoli isolates as valid character. It is an experienced fact that some isolates of M. phaseoli immediately develop sclerotia without having any mycelial stage, and thus it may be possible that this sclerotial stage of the isolates of this fungus might have failed to incite the infection. Considering this, it is felt necessary that patho- genicity of M. phaseoli still needs investigations by studying quite a large number of isolates, from a variety of plants in different localities of several countries.

BUTLER (1918) and LUTHRA & VASUDEVA (1938) described the mycelium of Rhizoctonia and gave the range of mycelial width. PRASAD (1944) reported that there were no differences in the des- cription and the mycelial width of the isolates from castor, cotton and sesame. In the present studies also, there were no differences in the description and width of mycelinm among the isolates and these were in agreement with those given by these authors.

In the present studies, also, there was not much difference in colour, structure and shape of the sclerotia, though sesame isolate formed abundant barrel shaped cells. The mycelium of the isolate was profusely branched with several lateral growths. Although all the isolates formed scletoria according to types I to IV described by REICHERT & t:h~LLINGER (1947), the common types observed in all the isolates were III and IV. The isolates of castor, groundnut and sesame had sclerotial diameter below 120 # thus falling in the group 'C' of HAIGH (1930). The sclerotial diameter of cotton isolate was, however, little more (128 #) than that proposed by HAIGH

260 N. B. K U L K A R N I ~ B. C. PATIL

for group 'C'. The statistical analysis of the data on the sclerotial diameter revealed that there was significant difference in the mean sclerotial diameter of all the isolates. These results are in conformity with the findings of WEST (1931) who stated that there might exist significant differences in the mean sclerotial diameter of M. phaseoli isolates belonging to group 'C'. There was no difference in the structure and shape of pycnidia, and pycnidiospores among the isolates. The details of the measurements of pycnidia and pycni- diospores of all the isolates from their hosts obtained as per technique developed by KULKARNI et al. (1962), indicate that cotton isolate had the smallest pycnidia 141 # (74--191 #), sesame and castor isolates intermediate [castor 162 # (98--250 #), sesame 164 # (115-- 239 #)] and groundnut isolate - - the largest 177 Ft (109--257 #). The differences among each group were significant. The pycnidial size was more or less the same when cotton isolate was inoculated on castor and groundnut (162 # on castor and 167 # groundnut). On basis of critical differences in spore length, there were two groups - - smaller group --- castor isolate 17.62 # (10.24--24.64 #), cotton isolate - - 17.73 # (9.41--23.19 #) and groundnut isolate 18.69 # (14.63--24.66#) and larger group - - sesame isolate - - 20.13# (11.23--30.51 #). The spore length was the same when cotton isolate was inoculated on castor and groundnut. Spore width was the smallest in castor isolate 5.48 # (4.59--6.27 #) and the largest in cotton isolate 5.99 # (4.80--7.10 #) and there was significant difference between the two. Groundnut isolate had 5.56 # (4.184 7.10#) and sesame isolate - - 5.63# (4.80--7.10#) spore width, which was non-significant when compared to that of castor or of cotton isolates. Further, pycnidia were developed without any stroma and were erumpent, globose, dark to grayish coloured hard bodies. Pycnidiospores were long, glassy, thin walled, elliptical and remained continuously hyaline even after germination. The ratio between length and width was always 3 : 1. None of the above authors could report these measurements for want of suitable technique in obtaining these structures on their respective hosts.

Sesame isolate differed from the other isolates in the production of aerial hyphae, colour of the colony and production of sclerotia. While the mycelial growth of sesame isolate was, in general, similar to sub-species 'Sesamica' of REICI~EtCT & HELLINGER (1947), its mycelinm was not slow growing, not so persistent, its colony not having concentric rings, but the isolate was pathogenic to bean. Remaining isolates agreed with their sub-species Intermedia; al- though the mycelium was not slow growing in cottolI and groundnut isolates. THIRUMALACHAR (1953) placed his castor isolate under sub- species 'Sesamica' on the basis of hyaline aerial hyphae when young but later turning smokey grey with concentric zones. In the present studies 'castor' isolate did not show such characters, but showed characters true to the sub-species 'Intermedia' of REICHERT & HELLINGER (1947). VERNEAR (1957) also found that his castor

NOS{t~NCLATURt~ OF MACROPIIOiMINA p t t A S E O L I 261

isolate belonged to 'Intermedia' sub-species. The classification of M. phaseoli proposed b y REICHERT & HELLINGER (1947) did not come true for sesame isolate in all the characters. Similarly the findings of THIRUMALACHAR (1953), about inclusion of castor isolate under 'Sesamica' sub-species also, did not agree with those now obtained.

Temperature growth relations of the isolates under s tudy gave interesting results. The opt imum temperature was between 30 ° and 85 ° C and agreed to the findings of VASlJDEVA (1986) and other workers. The castor isolate was slow growing, in general, with very poor development of sclerotia. Sesame isolate developed abundant sclerotia at all the temperatures. At 35°C sesame and groundnut isolates developed concentric rings, which clearly sup- ports the inclusion of sesame isolate, under 'Sesamica' sub-species. Groundnut isolate should also be placed under 'Sesamica' sub- species if this character alone is taken into account. I t however, differed in pathogenicity, being not slow growing and not persistant. I t is therefore, a matter of consideration whether groundnut isolate falls under 'Sesamica' or 'Intermedia' sub-species.

Carbon requirements including utilization of arabinose, raffinose, dulcitol, inositol, dextrin, ghlcose, lactose and sucrose, in general, were the same for all the isolates. In this respect also 'sesame' isolate was different from the other isolates in the amount of aerial hyphae, production of sclerotia and colour of the colony. MoNIz & BI~IDE (1963) considered utilization of lactose as one of the impor- tant physiological characters to delimit M. phaseoli into a variety, which fact, however, does not just ify in the light of the results obtained in the present studies. No differences in the utilization of amino compounds were observed among the isolates.

Summarising the results, it may be said that the taxonomy and nomenclature of M. phaseoli requires critical examination in the light of the findings of THIRUMALACHAR (1953), EDWARD (1954), HAIOH (1930), REICHERT & HELLINGER (1947) and those obtained in the present investigations. As already stated, the validity of the genus Macrophomina remains and all the isolates under s tudy were true to M. phaseoli species. Pathogenicity of M. phaseoli as discussed above requires critical examination taking into account the stage of the mycelium, technique of inoculation and the environmental conditions. REICHERT & HELLINGER (1947) classified M. phaseoIi into Sesamica, I~,termedia and Ty~ica, sub-species. The sub-species Sesamica was not in agreement with the sesame isolate so far as pathogenicity was concerned. In respect of other characters of Sesamica also, the mycelium of sesame isolate was never slow growing, but was dense, fumacious and had concentric zonation only at 35 ° C. The scterotial formation agreed to group I I I and IV of REICH~RT & HELLINGER as well as that of HAIGtI ('C' group). Sesame isolate produced abundant sclerotia and dark olive grey mycelium in most of the carbon and nitrogen compounds.

262 N. B. K U L K A R N I ~ D. C, PATIL

The castor isolate was slow growing with poor development of sclerotia in general, but was more or less true to the sub-species 'Intermedia' and not to 'Sesamica'. Cotton isolate was also agreeing in most of the characters of the sub-species 'Intermedia' except that mycelium was not slow growing. Groundnut isolate, however, was similar to cotton isolate in most of the characters except that it formed concentric zonations at 35 ° C and good amount of sclerotia in nitrogen compounds. Considering all aspects, it is felt that dis- tinct differences in certain characters exist amoung the isolates under study. MONIZ & BttlDE (1963) created a variety of M. flhaseoli from cotton as M. phaseoli var. Indica, on the basis of pathogenicity, slight morphological differences and variations irt cultural and physiological characters.

Considering the basic fact that M. phaseoli is a highly cosmopoli- tan fungus attacking a variety of plants under different climatic conditions, it is only natural that the differences and variations could be observed among the various isolates obtained from the same host. Unless a critical s tudy incorporating quite a large number of isolates obtained from different localities on the same host is undertaken, it is felt that it woMd be rather unwise to create a variety on the basis of the inadequate data at present available. Even the morphological characters viz. size of the pycnidiospores, that too obtained by M. phaseoli isolate on potato plant, although reported by MONIZ (1961) to be statistically significant, in the myco- logical studies, difference of a few microns in the size does not justify its inclusion in the character differences for creating a variety of such a cosmopolitan fungus. Besides this, the present s tudy reveals that no difference could be noticed in the mycelial or sclerotial development as in all the isolates the mycelium was persistent and fumacious. This character of mycelium was also taken as valid by MosrlZ & BHIDE (1963) in creating a variety. In the light of the facts stated above and also on the basis of results obtained, creation of a variety appears to be untenable unless supported by specific charac- ter differences in a large number of isolates. In the present studies, considering the taxonomic characters all the four isolates were true to the type species M. phaseoli and the validity of this binomial as suggested by YOUNG (1949) should remain without controversy.

For the identification of different isolates of M. phaseoli as has been done by REICHERT & HELLINGER by creating 3 sub-species and also b y HAIGH by creating 3 groups, it will be desirable to adopt the system followed by REICHERT • HELLINGER on a rational basis, combining the essential features of the findings of HAIGH as well as those obtained in the present studies. Thus the system of creating sub-species of M. phaseoli proposed by I~ElCVlERT & HELLINGER (1947) needs modifications particularly in mentioning therein detailed description of the asexual stage i.e. pycnidia and pycni- diospores. This system will only be complete when comparative s tudy of pycnidia, their size and the size of the pycnidiospores is

N O M E N C L A T U R E OF MACROPHOMINA PHA SEOLI 263

undertaken on a variety of hosts on which these stages are naturally formed or when produced by artificial inoculation by the pathogens. Until such s tudy is made it will be desirable to adopt the system proposed by these authors with the addition of one more sub- species for inclusion of the isolates of M. phaseoli whose sclerotia are upto 200/~ or more according to HAIGH'S B or A groups. It is beyond the scope of the present work to suggest such modified system as the number of isolates studied were only four.

According to the system of REICHERT & HELLINGER, the isolates under s tudy could thus fall under two sub-species as under - -

Sesame isolate 'Sesamica' s~lb-species

Castor, cotton and t 'Intermedia' sub-species. groundnut isolates J The inclusion of groundnut isolate under sub-species 'Intermedia'

has been made since it agrees in almost all the characters of this sub-species except one viz. it formed concentric rings at 35 ° C and that too only once. This however, needs further investigations.

Acknowledgements

The authors are thankful to Prof. M. SULAIMAN for the facilities given during this s tudy and to Prof. M. N. KAMAT for helpful suggestions.

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