[ 453 ]

Trans. s: mycol. Soc. 54 (3),453-460 (1970)Printed in Great Britain

FUNGAL INFECTION OF GROUNDNUTFRUIT BEFORE HARVEST

By D. McDONALD

Institute for Agricultural Research, Ahmadu Bello University,Samaru, Zaria, Nigeria

(With 1 Text-figure)

Fruits of the long-season groundnut (Arachis hypogaea L.), variety Samaru 38,were tested at intervals during development for fungal infection of the shell(endogeocarp) and of the seed. The shells were found to be infected by fungi atan early stage in development but infection of seeds did not take place untillater. The dominant fungi in the endogeocarpic mycoflora were Fusarium spp.and Macrophomina phaseoli; other common species included Aspergillus spp.,Botryodiplodia theobromae, Penicillium spp. and Rhizopus spp. Dominant fungi inseeds were Fusarium spp., Aspergillus spp. and Penicillium spp.

Many of the fungi which cause diseases of roots, pegs and fruits of thegroundnut, and which later may bring about spoilage of seeds duringpost-harvest drying and storage, are common soil inhabitants. One suchfungus, Aspergillus flavus, has been the centre of much interest in recentyears because of its ability to produce aflatoxins. In order to understandthe factors influencing the occurrence of A.flavus in the groundnut fruit thedevelopment of the fruit mycoflora from its earliest stages was studied. Ithas been reported by Wilson (1947) that the young groundnut fruit isoften invaded by soil fungi soon after it penetrates the soil. Garren (1966)indicated the existence of a fairly well defined endogeocarpic mycofiorawithin the fruit as it developed in the soil and suggested that normalsuccession led to predominantly healthy fruits with quiescent fungi, butthat disturbance of the succession could lead to rotting of fruits. In­vestigating the fungi developing on windrowed groundnuts in Georgia,Jackson (1965) obtained indications of the occurrence of a succession offungi in the drying fruit. Further investigations by Jackson (1968) onboth immature and mature fruits enabled him to distinguish severalseral communities and to observe progressive changes in the endogeo­carpic fungi.

Experiments were carried out during the 1965 and 1966 seasons atMokwa Agricultural Research Station, Nigcria, in which fungal infectionof fruits of the variety Samaru 38 was studied from an early stage in fruitdevelopment until harvest.

454 Transactions British Mycological Society

MATERIALS AND METHODS

The variety Samaru 38, which was used in both years, is a member ofthe Castle Cary Bunch Group and takes about 18 weeks to reach maturity.Pods are of medium size and contain, typically, two medium-sized seedswhich are dormant at harvest. On 2 June 1965 and on 4 May 1966, seedswere sown at 23 em spacing along ridges 91'4 em apart, one seed beingplaced in each planting hole.

Mokwa (9° 19' N; 5° 00' E) has an annual rainfall of 1000-1270 mmdistributed largely between I April and 31 Oct. The soil is classified as ared ferrisol on loose sandy sediments.

In both experiments the crop was laid out in twenty replicate plots tofacilitate sampling. At each time of sampling, one plant was lifted fromeach plot and the most mature pods detached. Pods from all plots weremixed together and samples taken for examination of shell and seedmycofloras.

In 1965, pods were first sampled 8 weeks after sowing while in 1966 thefirst sampling was done after 9 weeks. Further samples were taken atweekly intervals until harvest which was at 18 weeks after sowing in bothexperiments.

Shell and seed mycofloras were investigated as follows: (I) 1965: Fiftyundamaged, two-seeded pods were washed in running tap water for 5 minthen surface sterilized by immersing them for 3 min in a I % aqueoussolution of sodium hypochlorite. The pods were then rinsed in sterilewater and removed to a sterilized cabinet where they were split open andseeds and half shells transferred aseptically to the surface of Czapek Dox­rose bengal-streptomycin agar medium in Petri dishes (9 ern diam). Fiveseeds or five half shells were plated out in each dish. The half shells werearranged with their convex surfaces in contact with the culture medium.The plates were incubated at room temperature (27-32 "C) and examinedfor growth offungi after 4 days and again after 8 days. Fungi growing outfrom shell portions and seeds were identified and counted, unknownspecies being subcultured for further study. (2) 1966: The procedure forexamination of shells and seeds outlined above was followed except thatseeds, on removal from the surface sterilized pods, were themselvessurface sterilized before being plated out by immersion in a 0'5 %aqueous solution of sodium hypochlorite for 3 min and rinsing in sterilewater.

RESULTS

Shells

A total of 14 species of fungi were common to shells in both years; fivespecies were restricted to 1965, and ten species were found only in 1966.The percentages of shells infected by individual species at each time ofsampling are given in Tables I and 2 for 1965 and 1966 respectively.The more important fungi are also presented in Fig. I, in terms of theirpercentage composition of total shell fungi.

The percentage of shells infected increased from quite low levels at the

Groundnut infection. D. McDonald 455

beginning of the sampling period (Week 8, 1965; Week 9, 1966) to theregion of 95-100 % at lifting on Week 18. The increase was much morerapid in the second year.

From consideration of Tables I and 2 and of Fig. I, it is apparent thatthe same fungi were dominant in the shell mycoflora in both seasons.These fungi were, in order of decreasing abundance, Fusarium spp.,

Table I. The percentage of surface sterilized groundnut shellsinfected by various fungi in 1965

Percentage of shells infected by fungi,Weeks after sowing

-------"---------

Aspergillus flavipes(Bain. & Sart.)Thorn & Church

A, flavus Link ex Fr.Ai fumigatus Fres.A, nidulans (Eidam)Wint,

A, nigerTiegh.A, versicolor (Vuill.)Tirab,

Botryodiplodia theobromaePat.

Cylindrocarpon sp.Fusarium spp, 8Macrophomina phaseoli

(Maubl.) AshbyMonilia sp,Myrothecium verrucaria

(Alb. & Schw.) Ditm.Neocosmospora vasinfectaE. F. Smith

Paecilomyces varioti Bain,Penicillium citrinum ThornP. funiculosum ThornPhoma spp.Rhizoctonia solaniKuhnRhizopusspp.Dark sterile myceliaWhite sterile myceliaNo, of shells tested So

9

2

2

10

2

II 12

20

2

2

2

122

2

20

4

2

2

26

2

5°

16

2

2

2

2

4

5°

----,18

o'S

2 17'0

o'SI'S

IS'S1'0

S 7'0

o'S2'5

2

2I'S2'SI'SI'S

4 18'S

o'SSo 200

Macrophomina phaseoli, Aspergillus spp. and Botryodiplodia theobromae. Lessabundant, but still fairly common, were Penicillium spp. and Rhizopus spp.The fusaria were predominant except on Week 17 in 1965 and on Weeks 12and 14 in 1966 when they were replaced by M. phaseoli. In 1965 the fusariaformed a much higher percentage of total fungi than in 1966, but thereverse was true of the percentage of shells infected. M. phaseoli formedhigher percentages of the shell mycoflora and also infected a higherpercentage of shells in 1966 than in 1965. The aspergilli, particularlyA.flavus and A. niger, were also more abundant in shells in 1966.

Fusarium spp. were the most frequent invaders of shells during the earlystages offruit development but, although the percentage of shells they in­fected did not vary greatly as the fruit matured, their percentage composi­tion of total shell fungi tended to decrease with time. M. phaseoli tended

456 Transactions British Mycological Societyto increase both in number of shells it infected and in percentage composi­tion of total shell fungi as the fruit matured. Aspergillus spp. were virtuallyrestricted to the last 6 weeks of sampling, only appearing in shells of moreimmature fruits at Week 9 in 1966 when 2 % of the shell portions testedwere infected. Botryodiplodia theobrotnae occurred only in the last 4 weeks ofsampling in 1965 and in the last 3 in 1966. The penicillia were found only

Table 2. The percentage of surface sterilized groundnut shellsinfected by various fungi in 1966

Percentage of shells infected by fungi.Weeks after sowing.

,- '-------------,

9 10 II 12 16 18

Absidia sp.Aspergillus flatus Link ex Fr.A. nidulans (Eidam) Wint.A. nigerTiegh.A.fumigatus Fres.Bahusandhika caligans Batista &

Upadh.Botryodoplodia theobromae Pa t.Cephalosporium spp.Chaetomium sp. 2

Cunninghamella elegans Lendn.Fusarium spp. 4 4 1

Hendersonula toruloidea Nattr. 2

Macrophomina phaseoli (Maubl.) :) 2

AshbyMyrothecium indicum Rama RaoNeocosmospora tasinfecta E. F.

SmithPaecilomyces varioti Bain.Penicillium citrinum ThornP. ehrlichii Kleb.P. funiculosum ThomPhoma spp.Rhizoctonia solani K iihnRhizopus stolonifer (Ehrenb. ex

Fr.) Lind.Sclerotium rolfsii SaccoSyncephalastrum racemosum (Cohn.)

Schroer.Thielaoia basicola Zopf. 8 3White sterile myceliaNo. of shells tested 100 100

2

6 4 12 14 8 2

2G 3 12 2

2

662

2

2

4 1 3i 38 28 iO 48 44 484

38 10 60 16 36 3 8 34

4 4 2

2

2

6 2 2 2 2

6 6

4 24

4

2

4100 100 100 100 100 100 100 100

in the last half of the sampling period in 1965, but were spread moreuniformly in 1966. Penicillium funiculosum and P. citrinum were present inshells in both years, the former being the more abundant. Thielauia basicolaand Chaetomium spp. were found in shells tested in 1966 but were notisolated in 1965; both were present in the more immature fruits only.

Seeds

In 1965 no infection of seeds was found until the final sampling atWeek 18 when I %of seeds tested were infected by a species of Fusarium.

The percentages of seeds infected at different times of sampling in 1966

Groundnut infection. D. McDonald 457

1965 1966

.+. .+ .+.+.=:::::::::::::+.+ ••••••••• +.+•••••••+.+ • •••••••••••••

•••••••••••• +.+ .+ ••• +.+ •••••• • • •••• +.+ ••• + •••••••••••••• • •• •• • • + ••••• + •••••••••••••••• • •••••••• +.+.+ •••••••••••••••• + ••••••• +.+.+ ••• + ••••• + ••••••••••••• + ••• + ••• + •••••••••••••+ ••••••• + ••• +. +.+.+ ••• + ••••• + ••• I••• • • • • • •• • •• • • • +.+.+.+.+ ••••••••••• + . + ••••• +.+ • • • +.. .... ....... 200 /

0+. +. +.+.+.+.+.+.+.+.+.+.+.+.+.+. ~

•••• +.+.+.+.+.+.+.+.+.+.+ ••••••••• +.+.+.+ ••••••• +.+ ••• +.+ ••••••••••••••• +.+.+ ••• +.+ ••• + •••••••••+.+.+.+.+.+.+.+++.+++.++ ••••••••.•••••+•.•••+.•••+•••• ++++ •••••••• +.+ ••• •• + •••• +++ +++++++••.++.+++.+++.+.++.+++++++++::::::::::::::::+:::::::::+::::::::::::::: +.:::::::+:::::::::+ +

•• +

l!JIDlI!ill11Ii! !I !1IIM

~~... ...... . ..... . ..... . .... . ... .... ... ~

...... . .. .. .. .. ....... . .... . .

[:::::--=a

~ «jc- <:><==:==J

[:>~<_.~.~-J

I I

17 18I

16I

15I

14I

13

Aspergillusspp .

Penicillium spp .

Rhizopus spp.

I12

I I I J ~IL _L--L_.J---L_...L...---I_...L-_L--J

12 13 H 15 16 17 18 9 10 11Weeks from sow ing

~ Fusarium spp. ~~g~.i:~i::~i:~il

l "i\~[~.F:Y Macrophomina phaseoli

.mlliI Botryodiplodia theobromae

c=J Other fungi

I !

10 11I

9I

8

Fig . t • Selected species as percentages of total groundnut shell fungi .

458 Transactions British Mycological Societyare shown in Table 3. No infection was found at Weeks 9 and 11 and onlyI % of seeds were infected at Week 10. From Week 12 to Week 18 seedinfection fluctuated between 16 and 2 %, no trends being discernible.

Although multiple infection occurred in shells, more than one species offungus was never isolated from the same seed.

Table 3. The percentage ofgroundnut seeds infected by various fungi in 1966

Percentage of seeds infected by fungi.Weeks after sowing

rr: "- \

9 10 II 12 13 14 15 16 17 18

Aspergillus jlavus Link ex Fr. 3 2A.fumigatus Fres.A. nigerTiegh. 4Chaetomium spp. 5Fusarium spp. 7 6 7 3 3Macrophomina phaseoli (Maubl.)

AshbyPenicillium citrinum Thorn 3 2P.funiculosum Thorn 4 4Rhizopus arrhizus Fisch. 2R. stolonifer (Ehrenb. ex Fr.)Lind.

White sterile mycelia I

No. of seeds tested 100 100 100 100 100 100 100 100 100 100

DISCUSSION

Shell infection

The present findings on the shell mycoflora differ considerably fromthose of Garren (1966) and Porter & Garren (1968) working in Virginia,U.S.A. Garren found that Trichoderma viride and Penicillium spp. weredominant in the endogeocarpic (shell) mycoflora of undamaged pods,while Fusarium spp. were regarded as successional species of no importancein the climax community. He considered Aspergillusflavus to be a persistentspecies but of little quantitative importance in undamaged pods. A. nigerfollowed a similar pattern to A.flavus but was less abundant. The presenceof a 'natural barrier' to pathogenic invasion of undamaged pods wassuggested by Garren who pointed to the possible antagonistic and competi­tive reactions of T. viride, and possibly some penicillia, within the endo­geocarpic community. Further investigations by Porter & Garren (1968)showed Penicillium spp. to be predominant in the shell mycoflora, Tricho­derma spp., Chaetomium spp. and Fusarium spp. to be dominant groups andA. flavus to be a subdominant species.

Jackson (1968), in Georgia, U.S.A., investigating the endogeocarpicmycoflora of undamaged pods found Fusarium spp. and Penicillium spp. tobe dominant while Thielavia spp., Chaetomium spp., Rhizoctonia spp. andSclerotium spp. were subdominant groups.

In both Virginia and Georgia the penicillia were an important groupwhile the aspergilli were oflittle quantitative importance. The reverse wastrue of Mokwa. Trichoderma spp., a dominant group in Virginia and ofmuch less importance in Georgia, were not found in shells at Mokwa. As

Groudnut infection. D. McDonald 459most of the shell fungi are of common occurrence in groundnut soils inwidely separated parts of the world, the differences mentioned above aremore likely to be due to climatic variation than to qualitative differencesin the soil mycofloras. T. viride is known to be much more common intemperate soils than in tropical soils. The penicillia are favoured by lowersoil temperatures and the aspergilli by higher temperatures (Jensen, 193I )

Seed infection

In 1965 the absence of infection of developing seeds until the finalsampling at Week 18 was in accord with previous findings in Nigeria(McDonald & Harkness, 1964) and in the United States (Jackson, 1968).The seeds were not surface sterilized before plating out whereas they weretreated with 0'5 % sodium hypochlorite solution in 1966 when up to16% of seeds were infected by fungi while still immature. It seems un­likely that the difference was due entirely to the different techniques.Rainfall during the 10 weeks before harvest was low in 1966. The build-upof fungi in the shells occurred much faster in 1966 than in the previousyear and this may have influenced seed infection. Joffe (1968), in Israel,using o· I % mercuric chloride as a surface sterilant found it reduced thenumber of fungi isolated from seeds, but gave an apparent increase in seedinfection by specific fungi. This he attributed to a selective removal ofantagonists. It is unlikely that the sodium hypochlorite had any selectiveeffect, but it may have operated by removing or modifying fungalinhibitors present in the testa.

In the present study Fusarium spp. dominated the seed mycoflora withAspergillus spp. and Penicillium spp. as subdominants. A. flavus was themost frequently isolated of the aspergilli. Of the penicillia the mostcommon were P. funiculosum and P. citrinum, both toxigenic fungi. Nodefinite increase in seed infection by fungi was evident in 1966 althoughlittle infection was found before Week 12. This is of interest as Jackson(1965) in Georgia, while finding seeds to be fairly free from fungi untilmaturity, did obtain some evidence of an increase in the percentage ofseeds invaded during maturation. The list of fungi found by Porter &Garren (1968) in seeds in Virginia corresponds fairly closely with thoselisted by Jackson but is very different from the fungi which occurred atMokwa.

This present study showed that fungi were present in shells from anearly stage in fruit development and for some considerable time before anyseed infection occurred. Fungal infection of shells increased as the fruitsdeveloped, but no such trend was evident in seed infection within thelimits of the experiment. Fusarium spp. were predominant in shells andseeds. Macrophomina phaseoli was a dominant species in the shell mycoflora,especially towards the end of the growing season, but was only onceisolated from seeds. Aspergillus spp. and Penicillium spp. were presentin shells in both years of the experiment and were isolated from seeds in1966. A. flavus was the most commonly occurring species of Aspergillus inboth shells and seeds and there is little doubt that this important fungushas a close relationship with the groundnut fruit.

Transactions British Mycological Society

Thanks are due to the Director, Institute for Agricultural Research,Samaru, for permission to publish this paper. I wish to express my apprecia­tion to Dr Eva R. Sansome, a.B.E., and to Professor Margaret A. Keayfor advice and encouragement during this work.

REFERENCES

GARREN, K. H. (1966). Peanut (groundnut) microfloras and pathogenesis in peanut podrot. Phytopath. Z· 55, 359-367.

JACKSON, C. R. (1965)' Growth of Aspergillus flaous and other fungi in windrowedpeanuts in Georgia. Part VII. Trap. Sci. 7,27-34.

JACKSON, C. R. (1968). A field study of fungal associations on peanut fruit. Res. Bull.Unio, Ga Coil. Agric. Exp. Stn no. 26.

JENSEN, H. L. (1931). The fungus flora of the soil. Soil Sci. 31, 123-158.JOFFE, A. Z. (1968). Mycoflora of surface sterilized groundnut kernels. Pl. Dis. Reptr 52,

608.McDoNALD, D. & HARKKESS, C. (1964)' Growth of Aspergillus jlauus and production of

aflatoxin in groundnuts. Part IV. Trop, Sci. 6, 12-27.PORTER, D. M. & GARREN, K. H. (1968). An analysis of the endogeocarpic microflora of

peanuts in Virginia. Trop, Sci. 10, 100-106.WIl.SON, C. (1947). A survey of fungi associated with peg and seed rots of peanuts in

S. Alabama. Phytopathologr 37, 24·

(Acceptedfor publication I I November 1969)