Suppression of tuber rot of yam caused by Aspergillus niger with a yam Rhizobacterium

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1 African Crop Science Conference Proceedings Vol. 8. pp Printed in El-Minia, Egypt ISSN X/2007$ , African Crop Science Society Suppression of tuber rot of yam caused by Aspergillus niger with a yam Rhizobacterium R. T. AWUAH * & K. O. AKRASI Department of Crop and Soil Sciences, KNUST Kumasi, Ghana, * awuahrt@yahoo.com Abstract: A yam rhizobacterium (isolate ESI) was evaluated for ability to suppress yam tuber rot artificially induced by Aspergillus niger. A tuber was inoculated by placing 20 µl conidial suspension of A. niger into each of four cork borer wounds along its length. On drying, inoculation points were treated with a ESI nutrient broth (NB) culture. Controls in which NB alone and Topsin M were applied to inoculation points and that where only ESI was used were established. After 8 days in humidified transparent polyethylene bags, the weights and volumes the rotten yam tissues, if any, were determined. When the tuber was inoculated with A. niger, an average of 6 g of tissue with a volume of 6 cm 3 was rotten. These were significantly higher (P 0.05) than the 2.96 g (volume, 2.25 cm 3 ) and 2.25 g (volume, 2.25 cm 3 ) rotten tuber tissues obtained when inoculation points were, respectively, treated with ESI and Topsin M. Values associated with the rhizobacterium and the Topsin M treatments were not significantly different. The ESI also suppressed sporulation of the A. niger and persisted at inoculation points. The bacterial broth culture and the NB did not cause tuber rot. Key words: Biocontrol, rhizosphere bacteria, thiabendazole, yam rot fungi Introduction One major constraint to yam production worldwide is post harvest rot which may be either physiological (Noon and Colhoun, 1979, 1981) or microbial (Emehute et al., 1998). Bonire (1985) estimated microbial post harvest losses in yam at 40% while Okigbo and Ikediugwu (2000) indicated that between 20 and 39.5 % of stored tubers may be lost to decay. Microbial agents causing rot of yam are mainly fungi (Nwankiti and Arene, 1978; Ogundana and Dennis, 1981; Ikuton, 1983; Osai et al., 1996; Cornelius and Oduro, 1999) but some bacteria, notably Corynebacterium and Serratia spp. as well as Erwinia sp. (Nwankiti and Arene, 1978; Emehute et al., 1998) are also known to be involved in yam tuber rot. One indigenous method reported to be effective in reducing post harvest microbial losses of yam is the application of wood ash and palm oil to the cut surface of yam tubers (Oduro et al., 1991). The treatment either delayed or prevented rot caused by Aspergillus niger, three species of Penicillium and Rhizopus stolonifer. A combination of wood ash and any broad-spectrum fungicide like Benlate or Thiabendazole have been recommended as dips for protection of yam minisetts against rot (Otoo et al., 2001). The authors indicated that, although treatment with wood ash alone gave good results, control was much better where the fungicide was added to the wood ash. Thiabendazole and benomyl may also be used to control post harvest tuber rot but the results are most promising when the chemicals are applied not later than three days after harvest and not after the rot organism is wellestablished in the tuber (Ricci et al., 1978). Plumbley et al. (1984) also suggested control of yam tuber rot with imazalil following resistance of the causal agent, Penicillium sclerotigenum, to benomyl. These not withstanding, other non chemical approaches such as biological control must be explored. Though several reports indicate successful use of antagonistic rhizosphere bacteria (rhizobacteria) against plant pathogens (Baker, 1987; Loper, 1988; Axelrood et al. 1988; Loper and Lindow, 1993), little is known about similar studies on yam tuber rots. One such report by Sanusi and Ikotun (1995) however indicated that an isolate of Pseudomonas fluorescens, an inhabitant of the rhizosphere of cassava, could be useful in the control of rot of yam and of cassava minisetts. Recently, Akrasi (2005) also demonstrated in vitro inhibition of some yam rot fungi viz: Aspergillus niger, Fusarium solani, Rhizopus stolonifer, Curvularia lunata and an Aspergillus sp. by a yam rhizobacterium which was designated isolate ESI. Culture filtrate of the bacterium was also fungitoxic. This makes the bacterium a potential candidate for use in controlling microbial rot of yam. The objective of the present study was, therefore, to compare the effectiveness of the rhizobacterium ESI with Topsin M in the control of yam tuber rot caused by artificial inoculation with Aspergillus niger. Materials and methods Rhizobacterial and Aspergillus niger preparations. The rhizobacterium used in the study was isolated from a yam rhizosphere soil collected at Ejura in the Ashanti region of Ghana. It was designated isolate ESI and has been shown to possess antifungal property (Akrasi, 2005). The bacterium was stored in a refrigerator until needed. When required for use, a culture was streaked on a nutrient agar (NA) plate and grown in the dark for 24 hr at 28 ± 2 ºC. A single colony of the bacterium was mixed shaken with 10 ml sterile distilled

2 R. T. AWUAH * & K. O. AKRASI water and 10 ml aliquot of the bacterial suspension mixed Biopsy of inoculated tissue samples for A. niger and ESI. with 100 ml NB and grown in the dark as above for 72h Recovery of ESI and A. niger from inoculation points was before use. attempted by excising tissues from such tissues, surface Aspergillus niger, was cultured on chloramphenicol (500 ppm) potato dextrose agar (CPDA) for 72 hr on a laboratory bench. Conidia of the fungus were suspended in sterilizing them in 10% commercial bleach solution (Eau de Javel containing 12 % chlorine) for 2 min and blotting them dry with tissue paper. To recover ESI, tissue bits were sterile distilled water in a 25-ml capped glass vial. The plated on PDA:NA medium (half strength PDA:half amount of conidia in the suspension was estimated using a haemocytometer to be 10 6 conidia/ml. Inoculation of yam tuber with A. niger and treatment with ESI. Yam (Dioscorea rotundata, Poir var. Puna) tubers (3-4 months old) were obtained from the market, thoroughly washed with water and left to dry. They were strength NA; 1:1 ratio) plates and incubated in the dark for 3 days at 28 ± 2ºC. For A. niger, tissues were plated on CPDA plates and incubated on a laboratory bench for 3-5 days. Five tissue bits per each inoculation point on a tuber (20 per tuber) were plated. Tissues were observed for either ESI or A. niger and the frequency of observation noted. surface sterilized by swabbing with 70% ethanol. Four 7 mm-diameter, 1 mm-deep cork borer wounds were made at Results 10-cm intervals along the length of each tuber. A 20 µl aliquot of the A. niger suspension was placed into each of All four sites on a tuber inoculated with A. niger became the four wounds (representing four replicate inoculations). diseased and soft after 8 days. In general, severity of rot On drying, each of the four inoculation points was treated increased from the tail region to the head region of the with 20 µl of the 72 h old NB culture of the rhizobacterium, tuber. A mean weight of 6 g rotten yam tissue was recorded ESI. The tuber was placed in a humidified transparent in the tuber inoculated with A. niger. This was significantly polyethylene bag and incubated in a dark incubator (37 ± different from the value of 2.96 g (representing 50% of 1ºC) for 8 days. It was then transversely cut at the points of suppression) obtained when the sites were inoculated with inoculation and the rotten tissues carefully removed with a A. niger and treated with ESI (Table 1). Rot suppression knife and separately weighed. In addition, the volume of obtained with the ESI was similar to that recorded for each rotten tissue was ascertained by placing it in a 50 ml Topsin M. distilled water contained in a 100 ml measuring cylinder. The volumes of rotted yam tissues paralleled the The volume of water displaced was recorded as the volume recorded weights of rotted tissues. Inoculation with A. niger of the rotten yam tissue. The intensity of sporulation by A. alone resulted in a mean tissue rot volume of 6.0 cm 3 while niger at inoculation points was qualitatively assessed. yam tuber inoculated with A. niger and treated with ESI Tubers inoculated with A. niger and treated with 0.02% gave a significantly lower mean rot volume of 2.95 cm 3 suspension of Topsin M (Thiophanate methyl; 70WP) as representing 50.3 % suppression in rot. Inoculation of a well as those inoculated with only the A. niger suspension, tuber with A. niger followed by treatment with Topsin M the ESI NB culture and NB alone were maintained as gave a mean rot volume of 2.25 cm 3 also representing controls. % suppression in tuber rot (Table 1). In all cases, the effects The weights and the volumes of the rotten yam tissues of the bacterium and the Topsin M in suppressing tuber rot were analyzed for differences between treatments using were not significantly different. Similar trends were MSTAT C statistical package. The experiment was obtained when the experiment was repeated. repeated. Table 1. Suppression of yam tuber rot with the yam rhizobacterium, ESI 1 Treatment 2 Wt of rotten tissue (g) 3 Vol of rotten tissue (cc) Sporulation 3 A niger alone 6.00 (-) 6.00 (-) +++ A. niger + ESI 2.96 (50.7) 2.95 (50.8) ++ Experiment 1 A. niger + Topsin M 2.25 (62.5) 2.25 (62.5) + ESI alone 0 (-) 0 (-) - NB 0 (-) 0 (-) - LSD A niger alone (-) (-) +++ A. niger + ESI 1.10 (97.00) 1.83 (95.2) ++ Experiment 2 A. niger + Topsin M 0.76 (97.80) 0.95 (97.5) + ESI alone 0 (-) 0 (-) - NB 0 (-) 0 (-) - LSD Each value is the mean of four replications. Values in parenthesis represent percent rot suppression. (-) = Not calculated because values are for non pathogen controls. 2 Treatments at inoculation points. 876

3 3 Intensity of sporulation of A. niger at inoculation points. +++ = very intense; ++ = moderate; + = sparse; - = none. Sporulation of A. niger was profuse at inoculation points where neither the bacterium nor Topsin M was applied and there was very little or no sporulation where the two protectants were applied to the inoculation points (Table 1; Fig 1). Fig. 1.Sporulation of Aspergillus niger at inoculation points on yam tubers. Top tuber, A. niger withoutesi NB culture treatment; Lower tuber, A. niger with ESI treatment. Note the abundant sporulation of A. niger at points where ESI was not used. Aspergillus niger was detected at all inoculated points where only the fungus was used. It was isolated at a frequency of 30% when the tubers were inoculated with A. niger and treated with ESI (Table 2). The fungus, however, was not detected where an inoculated yam tuber was treated with Topsin M (Table 2). Table 2. Recovery (%) of Aspergillus niger (AN) and ESI from inoculation points on yam tubers Inoculation sites 2 Treatment 1 I II III IV Experiment 1 AN ESI AN ESI AN ESI AN ESI A niger alone 100(5/5) 0(0/5) 100(5/5) 0 (0/5) 100(5/5) 0(0/5) 100(5/5) 0(0/5) A niger + ESI 60(3/5) 40(2/5) 0(0/5) 80(4/5) 60(3/5) 80(4/5) 0(0/5) 100(5/5) A niger+topsin M 0(0/5) 0(0/5) 0(0/5) 0 (0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) ESI alone 0(0/5) 100(5/5) 0(0/5) 100(5/5) 0(0/5) 100(5/5) 0(0/5) 100(5/5) NB 0(0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) Experiment 2 A niger alone 100(5/5) 0 (0/5) 100(5/5) 0 (0/5) 100(5/5) 0(0/5) 100(5/5 0(0/5) A niger + ESI 100(5/5) 60(3/5) 100(5/5) 60 (3/5) 100(5/5) 40(2/5) 100(5/5 60(3/5) A niger+topsin M 0(0/5) 0 (0/5) 0(0/5) 0 (0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) ESI alone 0(0/5) 100(5/5) 0(0/5) 100(5/5) 0(0/5) 80(4/5) 0(0/5) 100(5/5) NB 0(0/5) 0 (0/5) 0(0/5) 0 (0/5) 0(0/5) 0(0/5) 0(0/5) 0(0/5) 1 Treatments at inoculating points. 2 Figures in parenthesis indicate the number of yam tissues yielding either A. niger or ESI/total number of pieces plated. Discussion When yam tubers were inoculated with A. niger, considerable rot occurred confirming previous reports

4 R. T. AWUAH * & K. O. AKRASI (Ikotun, 1983; Adimora et al. 1989) that A. niger is a yam tuber rot fungus. Severity of rot increased from the head to the tail region of the tuber. This observation supports a report by Coursey (1964) indicating that the head region of the tuber is less susceptible to rot than the middle and end region. Emehute et al. (1998) suggested that variation in the water contents of the various tuber regions, with the head having the least, could be responsible for this. Arinze (1989), however, attributed the observed differences in susceptibility of the various yam portions to rots to variable distribution of phytoalexins within the head, middle and tail regions of the tuber. With the procedure used, the NB culture of ESI and the fungicide Topsin M were consistently similar in their effects against A. niger on yam tubers. This is desirable because it shows the potential of the rhizobacterium for development into a biofungicide. This result is consistent with a report by Di Petro et al. (1992) indicating that the antifungal antibiotic, Chaetomin, produced by Chaetomium globosum which suppresses Pythium ultimum) is comparable in effect to that of the fungicide metalaxyl. Suppression of conidia production by the ESI NB culture on yam tubers is a very significant result from the stand point of plant disease epidermiology since the amount of conidia produced by a pathogen reflects its inoculum potential (Bilgrami and Dube, 2001) and consequently the magnitude of the resulting disease (Agrios, 1997). Similar results by Sharma et al. (1996) showed that metabolites from antagonistic strains of Trichoderma and Gliocladium spp. reduced sporidia production in Tilletia indica. What is unclear in the current study is whether or not the NB culture of ESI could have protected the yam if it had been applied before inoculation of the tuber with A. niger. This needs to be verified. It is also necessary to prove efficacy of the ESI in traditional yam storage barns because good performance by a potential biocontrol agent in a controlled environment such as what pertained in the current study is not necessarily indicative of its ability to exert biocontrol activity under real life situations. In a real life situation, survival and colonization by the biocontrol agent and competition with other organisms in a dynamic environment (Parke, 1990) as well as the environmental conditions are factors that may influence successful biocontrol with a selected organism. Acknowledgment Partial funding provided by the International Institute of Tropical Agriculture (IITA), Ibadan for the conduct of this research is acknowledged. References Adimora, L.O., Oduro, K.A. & Damptey, L.O..A Studies of causal agents of rot in Dioscorea rotundata Poir vars. Gboko (White Yam). Ghana Journal of Science, 29-30, Agrios, G.N Plant pathology. 4th ed. Academic Press. 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