[ 445 ]Trans. Br. mycol. Soc. 54 (3), 445-451 (1970)
Printed in Great Britain
FUNGI ASSOCIATED WITH SOFT ROT OF YAMS(DIOSCOREA SPP.) IN STORAGE IN NIGERIA
By S. K. OGUNDANA, S. H. Z. NAQVI ANDJ. A. EKUNDAYO
School of Biological Sciences, University of Lagos, NigeriaThe organisms which cause soft rot of yams (Dioscorea spp. ) in storage are
identified as Botryodiplodia theobromae Pat., Fusarium moniliforme var. subgluti-nans Wr. & Rg. , Penicillium sclerotigenum Yamamoto, and Aspergillus niger vanTiegh, The fun gi ent er through wounds and natural openings on the surfaceof the tubers. Optimum rot by the pathogens occurs a t 90 %r. h. and at 26-30"C. The extent of rotting vari es with the fungus and the yam species.
Coursey (1961) attributed rotting by microbial action as one of thecauses of losses during storage of yams (Dioscorea spp.),
Bunting (1924), Dade & Wright (1931) and Piening (1962) associatedyam soft rot with Botryodiplodia theobromae. An anonymous report (1939)mentioned that soft rot in Chinese yam was caused by Fusarium OXY-sporum. Yamamoto, Yoshitani & Maeda (1955) attributed the rot ofChinese yam to F. solani, F. oxysporum and Penicillium sclerotigenum. Baudin( 1956) recorded Rhizopus nodosus as causing soft rot in yam while Miege(1957) established that B. theobromae, Lasiodiplodia sp. and Fusarium sp. alsocaused this disease. Okafor (1966) reported that B. theobromae, Penicilliumspp ., Macrophomina phaseoli, Hendersonula toruloidea, and a motile gram-negative, orange-red pigm ented bacterium, probably Serratia sp., alsocaused rot in yam tubers.
In the present study, the microorganisms responsible for soft rot inDioscorea rotundata Poir., D. alata L., D. cayenensis Lam. and D. dumetorumPax in storage were isolated and identified. The mode of entry of th epathogens as well as the environmental conditions enhancing the rotdevelopment were investigated.
MATERIALS AND METHODS
To isolate the causal organisms, diseased yam tubers were surfacesterilized and cut with a sterile knife starting from the side away from therot. From the advancing edge of the diseased areas, slices, 3 x 3 x 2 mm,were cut and plated on 2 % malt extract agar and incubated at 28.Since frequently the cultures obtained contained more than one fung alcolony subcultures were made until pure cultures were obtained by suc-cessive hyphal tip transfers.
To establish whieh of the isolates caused yam rot , cylindrical cores,2 ern long, were removed with a 6 mm diam sterile cork-borer from themiddle portion of eaeh freshly harvested ya m tuber. Disks (5 mm) of
Transactions British Mycological Society7-day-old fungal cultures, on malt extract agar, were placed into the holesin the tubers, and the cores of yam originally removed were replaced after3 mm pieces had been cut off to compensate for the thickness of the agarinoculum. The replaced cores were then sealed with vaseline underaseptic conditions. Disks of uninoculated malt extract agar were used ascontrols. All treated yam tubers were left in a yam barn under naturalconditions for 4 weeks. Rot was assessed to have occurred where themaximum distance of the growth of fungus from the site of inoculation asevidenced by the soft rot texture, in the inoculated yam, exceeded themaximum distance ofcut tissues in the uninoculated yams. Rot was alwaysconfirmed by microscopic examination of the inoculated tubers for thepresence of the pathogen.
To determine the effect of temperature, peeled yam slices about10 x 10 X 50 mm were surface sterilized, inoculated with the mycelialsuspension of the different fungi and placed in sterile McCartney bottles.Slices were incubated for 2 weeks at 10, 20, 26, 30, 35 and 40 and ex-amined for rot as described above. To test the effect of relative humidity,inoculated yam disks were put on sterile copper wire gauze in the upperchambers of desiccators, the lower chambers of which contained 250 ml ofsaturated salt solutions having an excess of the solid phase of each of thesalts used to give the desired relative humidities of 15, 45, 80 and go %.Sterile distilled water was used to obtain 100 %r .h, The desiccators wereplaced in a constant temperature chamber at 25 for 2 weeks after whichthe yam slices were examined for rot.
Symptoms ofsoft rot inyamsThe rotting of the tubers of the four species of yam examined generally
commenced at some point ofwounding. Early symptoms included soft darkspots, roughly circular or irregular in outline but with edges that tended tobreak away from the healthy tissue. With D. dumetorum the decay wassuperficial, involving the' skin' and a thin layer of cell s beneath it. Some-times the rot occupied most of the interior of the tuber. As the decayprogressed, water-soaked lesions occurred.
The diseased tissue exposed after cutting through a tuber was usuallybrown and as the disease progressed the regions first infected turneddarker. Other colourations occurred depending on the yam species andthe type of pathogen, for instance, blackening occurred when D. rotun-data was infected by Aspergillus niger and a dirty grey tissue was formedwhen the pathogen was B. theobromae. Fusarium spp. showed purple toyellowish pink colourations on D. dumetorum.
Isolation and incidence of rotting inyamTo isolate the causal organisms of soft rot of yams, tubers of D. roiun-
data, D. alata, D. cayenensis and D. dumetorum showing symptoms of diseasewere employed. Small pieces of diseased tissue were removed asepticallyand plated on malt extract agar. The following fungi were isolated:
Soft rot ofyams. S. K. Ogundana et al. 447B. theobromae Pat.; F. moniliforme var, subglutinans Woron. & Reinking;F. poae (Peck) Woron; F. solani (Mart.) Sacc.; F. coeruleum (Lib.) Sacc.;Mucor circinelloides van Tiegh.; R. stolonifer (Ehrenb. ex Fr.) Lind.; P.sclerotigenum Yamamoto; Trichoderma viride Link ex Fr.; Gliomastix convulata(Harz.) Mason; A. niger van Tiegh.; Gliocladium roseum Bain.; Rhino-cladiella mansonii (Castell.) Schol-Schwanz.
To determine the incidence of rotting in the four different yam species,10 rotten tubers of each species were examined for the types of pathogenspresent. Results are given in Table I which shows that B. theobromae,F. moniliforme, P. sclerotigenum, A. niger and R. stolonifer were isolated frommore than half of the yam tubers while the other fungi were isolated onlyoccasionally. All the yam species tested were susceptible to F. monili-forme and P. sclerotigenum. D. alata appeared to be the most susceptible toP. sclerotigenum.
Table I. Fungi in rotten yamsNumber of yarns affected*
D. rotundata D. alata D. cayenensis D. dumetorumB. theobromae 7 10F. monilijorme 6 2 2 4F. poae I IF. solani 2 IF. coeruleum IM. circinelloides I 2R. stolonifer 3 2P. sclerotigenum 8 7 10 3T. viride 2 2G. convulata IA. niger 4 2 2G. roseum 2R. mansonii I
* Isolates were made from at least 6 different sites on each yam.10 tubers of each species were employed.
The isolates were reinoculated into yam tubers and their ability tocause rot was assessed by measuring the distance of rot from point ofinoculation. Table 2 illustrates the maximum distances of mycelial growthof the fungi in the tubers from the point of inoculation after 4 weeks.
The most severe rot-causing organisms were B. theobromae, F. moniliforme,P. sclerotigenum and A. niger. F. poae, F. solani, F. coeruleum, T. viride andG. convulata caused only mild rotting and were therefore considered to beweak pathogens. Mucor circinelloides, R. stolonifer, G. roseum and R. mansoniicaused no rotting.
Mode ojfungus infectionTo study the mode of entry of the fungi mycelial suspensions of 7-day-
old cultures were sprayed on wounded and unwounded yam species whichhad been surface sterilized with absolute ethanol and washed with steriledistilled water. Tubers were wounded by exposing with a scalpel a fairlylarge area near the surface of the tubers. Ten tubers of each of the threeyam species-D. rotundata, D. alata and D. cayenensis-which showed rotsymptoms during earlier experiments were employed. Each tuber was
448 Transactions British Mycological Societyplaced in a sterile polythene bag for 4 weeks on a rack near the yam barnunder natural weather conditions. Unsprayed wounded and unwoundedyam tubers were used as control. The yam tubers were then examined toestablish the extent of rot.
Table 2. Maximum distance ofgrowth (mm) offungi in inoculated yam tubersafter 4 weeks ofincubation in the yam-barn under natural conditions
Maximum distance of growth (mm), --"
D, rotun- D, cayen- D, dume-data D, alata ensis torum Remarks
B, theobromae 28'0 11'0 29'7 5'2 Rotting*F, moniliforme 22'0 14,6 19'0 17'0 Rotting
F, poae 5'5 4'4 3'2 0'4 Mild rottingF, solani 6'5 5'3 4'5 1'0 Mild rottingF, coeruleum 4,8 1'2 3'5 3'0 Mild rottingM, circinelloidea 0'0 1'0 0,8 1'2 No rottingR, stolinifer 2'5 2'0 1'5 0'7 No rotting
*P, sclerotigenum 34'0 15'2 4 2'2 24'5 RottingT, viride 4'5 3'0 4'5 2'0 Mild rottingG, convulata 3'0 2'5 2'5 2'0 Mild rotting
*A, niger 38'0 24'5 4 0'1 10'0 RottingG, roseum 2'0 1'5 1'5 1'3 No rottingR, mansonii 2'0 1'0 1'3 2'0 No rottingControl 2'5 1'5 2'0 1'2 No rotting
The distances expressed above are the averages of 10 measurements,* Isolates that caused appreciable rot,
Table 3. Maximum distance of growth (mm) offungi inoculated into yamsthrough cuts and wounds
Wound CutB, theobromae 20 21 16 II 26 20F. moniliforme 10 14 9 10 10 12P. sclerotigenum 25 23 5 13 36 29A, niger 30 27 14 10 3 35
These readings are the averages of 6 measurements,
Mycelial growth was observed externally on all the tubers which had beeninoculated. This occurred on the wounds created by peeling or scraping.Mycelial growth was also observed both at the points where there hadbeen some rootlets, and sometimes at the lacerations on the tubers. Thetubers were cut into pieces through the wounds and the rot assessed.Table 3 shows the extent of rotting from the site of wounds due to peelingor scraping and 'cuts'. There was no rot in any of the tubers used as acontrol.
These results suggest that the penetration of the rot-causing organismstakes place through wound or cuts created as a result of mechanicalinjury during harvesting or in transit.
Soft rot ofyams. S. K. Ogundana et al. 449Effect of environmental factors on the development of rot
The effect of temperature on the ability offungi to infect yam and causerot was investigated as described in Materials and Methods. Results areshown in Table 4.
In the two yam species used, rot occurred over the range of 20-30 forall the pathogens. B. theobromae and A. niger caused some rot at 35, whilstP. sclerotigenum caused slight rot at 10. Invariably optimum rot occurredbetween 26 and 30.
The results of investigations into the effect of relative humidity on theability of the isolates to cause yam rot are shown in Table 5. These indicatethat there was little or no rot at 15 % r.h., while some rot of some yamspecies by A. niger occurred at 45 %r.h. Appreciable rot of all yam speciesby the pathogens occurred at r.h. above 80 %.Table 4. The eifect of temperature on maximum distance of growth (mm) of
tuber rot caused by the pathogens in two yam species(i) Dioscorea rotundata
Temperature (0C)-' -----,
10 20 26 30 35 40B, theobromae 0 3 4 5 2 0F, moniliforme 0 2 2'5 3 0 0P. sclerotigenum 2 5 7 6 0 0A, niger 0 4 5 4'5 3 0
(ii) Dioscorea caynensisTemperature (0C)
10 20 26 30 35 40B, theobromae 0 3 4 4'5 2 0F, moniliforme 0 2 3 3 0 0r. sclerotigenum I 2'5 4'5 4 0 0A, niger 0 2'5 4- 5 2 0
Apart from the metabolic processes such as sprouting and respiratoryactivities, and loss of moisture content by evaporation, storage rots arethe main factors responsible for the deterioration of stored yams (Coursey,1967). In the present study the micro-organisms that caused appreciablerot in tubers were found to be Botryodiplodia theobromae, Penicillium sclero-tigenum, Aspergillus niger, Fusarium moniliforme while F. solani, F. poae, F.coeruleum, Trichoderma viride and Gliomastix convulata seemed to be weakpathogens. Probably the most important of these is the ubiquitousorganism B. theobromae, which has been shown by Dade & Wright (1931)to be associated with at least three apparently quite distinct forms of tuberrotting.
This is apparently the first time that A. niger, F. moniliforme, F. poae, T.viride and G. convulata have been reported to be associated with yam rot,with only A. niger causing appreciable rot.
29 M YC 54
Table 5. Effect ofrelative humidity onrot development infoury am species expressed as themaximum distance of growth (mm) f rom the site of inoculation
r .h ,15 % r, h 45 % r .h.80 % Lh 90 % r. h . 100 %~---., , -' ---, , A ---, ,-- A ---, ,----"------,*R A C D R A C D R A C D R A C D R A C D
B. theobromae I - 1'5 1 6 4 4 2 6 5 4 3 10 6 5 3' 5 7 5 4 2'5++ + + - + + + + + + +++ + + ++ - ++ + + +
P. sclerotigenum 1 1'5 2 2 4 4 1 2 5 4 1 1 6 6 4 5 6 4 2 3+ + - - + + + - - ++ ++ + ++ + + + - +
F. moniliforme 1 1 0'4 0'5 3 5 3 2'5 4 5 4 2 5'5 6 7'5 3 3 6 4 4++ + - + + + + - ++ + + + + - - + + + +
A, niger 2 - 0'5 0'5 4 6 8 2'5 4'5 6 9 3 6 10 14 4 5 9 10 4+ ++ + + - + + + + + + + + + + + + ++ + + + + + + ++ +
- = N o h yphae obse rved ; + = little rot ; + + = appreciable rot ; + + + = ex tensive rot ... Key to yam spec ies: R = D . rotundata; A = D . ala/a ; C = D . cayenensis; D = D . dumetorum.
Soft rot of yams. S. K. Ogundana et aI. 451The pathogens responsible for the soft rot enter the host through
wounds and mycelial growths developed at natural openings like the scarleft by the rootlets and lacerations on the tubers; no fungal penetrationsoccurred through the unwounded surfaces of the yam tubers. This indi-cates that, apart from any natural openings, mechanical damage to tubersat harvesting can permit infection by the rot fungi. Further bruising ofthe tubers may also occur during collection and packing on the farm andduring subsequent transportation and storage.
The indications are that rotting in storage can be prevented by re-ducing surface damage to the tubers during harvesting and transportationto the place of storage.
ANONYMOUS (1939). Rep. P.R. agric. Exp. Stn. 1938, p. 39.BAUDIN, R. H. (1956). Maladies parasitaires des ignames en Cote d'Ivoire. Revue Mycol.
21, 87-1 I 1.BUNTING, R. H. (1924). Appendix A. Report of the Research Branch rst January 1922 to
31st March, 1923. Rep. Dep, Agric. Gold Coast, 1923, p. 20.COURSEY, D. G. (1961). The magnitude and origins of storage losses in Nigerian yams.
J. sci. Fd. Agric. 12, 574-580.COURSEY, D. G. (1967). Yams. An account ofthenature, origins, cultivation andutilisation ofthe
useful members ofthe Dioscoreaceae. London: Longmans, Green and Co. Ltd.DADE, H. A. & WRIGHT J. (1931). n. Gold Coast, Dep. Agric. 1930, pp. 249-250.MIEGE, J. (1957). Influence de quelques caracteres des tubercules-semences sur les
levees et le rende...