postharvest biological control of anthracnose (colletotrichum gloeosporioides) on mango (mangifera...

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Postharvest Biology and Technology 50 (2008) 8–11 Contents lists available at ScienceDirect Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio Postharvest biological control of anthracnose (Colletotrichum gloeosporioides) on mango (Mangifera indica) Yonas Kefialew a , Amare Ayalew b,a Gambella Agricultural Research Institute, P.O. Box 62, Gambella, Ethiopia b Department of Plant Sciences, Haramaya University, P.O. Box 241, Haramaya, Ethiopia article info Article history: Received 2 September 2007 Accepted 15 March 2008 Keywords: Antagonistic yeast Antagonistic bacteria Colletotrichum gloeosporioides Mango anthracnose Postharvest biological control abstract Preliminary screening of fungi and bacteria isolated from unmanaged mango trees in different ecolo- gies of Ethiopia, yielded isolates antagonistic towards Colletotrichum gloeosporioides, the cause of mango anthracnose. Four isolates of bacteria, five yeasts and two filamentous fungi were evaluated in this study. Cell suspensions and culture filtrates of the isolates inhibited spore germination and hyphal growth of C. gloeosporioides in vitro. The isolates significantly reduced severity of anthracnose on artificially inoculated mango fruit. Brevundimonas diminuta isolate B-62-13, Stenotrophomonas maltophilia L-16-12, a member of Enterobacteriaceae L-19-13, Candida membranifaciens F-58-22, and the yeast isolate B-65-23 which, based on ITS analysis, possibly represents an undescribed species, were effective on naturally infected fruit. They kept anthracnose severity (lesion development) below 5% during much of the 12d experimental period while severity on untreated fruit reached 29%. B. diminuta and the yeast B-65-23 were as effec- tive as hot water treatment at 55 C for 5 min. Further investigations on the mechanisms of biocontrol involved and the safety of the isolates, particularly the bacteria, for use on edible fruit are warranted. Only a single application of the isolates showed a potential for the control of mango anthracnose on harvested fruit. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Anthracnose, caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc., is the major postharvest disease of mango in all mango producing areas of the world (Dodd et al., 1997). The disease occurs as quiescent infections on immature fruit and the damage it incites is more important in the postharvest period (Muirhead and Gratitude, 1986; Dodd et al., 1997). Fungicides, either as preharvest or postharvest treatments, form the main approach to reduce losses from anthracnose. However, their use is increasingly restricted due to public concerns over toxic residues. Moreover, fungicides are unaffordable for many mango growers in developing countries (Dodd et al., 1989). Postharvest control of mango anthracnose could also be accomplished by treat- ment of fruit in hot water, alone or in combination with chemicals (Dodd et al., 1997). Precise control of temperature and time is criti- cal, as fruit can be easily damaged by overexposure to heat (Arauz, 2000). Effective biocontrol agents offer great potential to develop alternative methods that are economical and suited for adop- Corresponding author. Tel.: +251 25 5530331; fax: +251 25 5530316. E-mail address: [email protected] (A. Ayalew). tion by the small-scale mango industry. However, few biocontrol agents have been reported against postharvest diseases of tropical fruit. Among the limited isolates, a nonpathogenic strain of Col- letotrichum magna (Prusky et al., 1993), isolate 558 of Pseudomonas fluorescens (Koomen and Jeffries, 1993), a strain of Bacillus sp. (De Jager et al., 2001), Bacillus licheniformis (Govender and Korsten, 2006), and preharvest application of the yeast Rhodotorula minuta (Pati ˜ novera et al., 2005) have been found effective for the control of postharvest diseases of mango. This paper reports on the antagonism of microorganisms initially isolated from mango towards C. gloeosporioides in vitro and the potential of isolates of Brevundimonas diminuta, Stenotrophomonas maltophilia, an Enterobacteriaceae, Candida mem- branifaciens, and a possibly new yeast species for the control of anthracnose development on harvested mango fruit. 2. Materials and methods 2.1. Isolates of antagonists and the pathogen The potential biocontrol agents were selected from a screening of 608, 563 and 402 isolates of bacteria, yeasts and filamentous fungi, respectively. They were initially isolated from mango leaves, blossoms and fruit sampled from unmanaged trees in different 0925-5214/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2008.03.007

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Page 1: Postharvest biological control of anthracnose (Colletotrichum gloeosporioides) on mango (Mangifera indica)

Postharvest Biology and Technology 50 (2008) 8–11

Contents lists available at ScienceDirect

Postharvest Biology and Technology

journa l homepage: www.e lsev ier .com/ locate /postharvbio

Postharvest biological control of anthracnose (Colletotrichum gloeosporioides)on mango (Mangifera indica)

Yonas Kefialewa, Amare Ayalewb,∗

a Gambella Agricultural Research Institute, P.O. Box 62, Gambella, Ethiopia

fungiolateof bare fil

e isolas dim, Can

repreverituntrent atthe isisola

b Department of Plant Sciences, Haramaya University, P.O. Box 241, Haramaya, Ethiopia

a r t i c l e i n f o

Article history:Received 2 September 2007Accepted 15 March 2008

Keywords:Antagonistic yeastAntagonistic bacteriaColletotrichum gloeosporioidesMango anthracnosePostharvest biological control

a b s t r a c t

Preliminary screening ofgies of Ethiopia, yielded isanthracnose. Four isolatesCell suspensions and cultugloeosporioides in vitro. Thmango fruit. BrevundimonEnterobacteriaceae L-19-13on ITS analysis, possiblyThey kept anthracnose seperiod while severity ontive as hot water treatmeinvolved and the safety ofa single application of thefruit.

1. Introduction

Anthracnose, caused by Colletotrichum gloeosporioides (Penz.)Penz. and Sacc., is the major postharvest disease of mango in allmango producing areas of the world (Dodd et al., 1997). The diseaseoccurs as quiescent infections on immature fruit and the damage itincites is more important in the postharvest period (Muirhead andGratitude, 1986; Dodd et al., 1997).

Fungicides, either as preharvest or postharvest treatments, formthe main approach to reduce losses from anthracnose. However,their use is increasingly restricted due to public concerns over toxicresidues. Moreover, fungicides are unaffordable for many mangogrowers in developing countries (Dodd et al., 1989). Postharvestcontrol of mango anthracnose could also be accomplished by treat-ment of fruit in hot water, alone or in combination with chemicals(Dodd et al., 1997). Precise control of temperature and time is criti-cal, as fruit can be easily damaged by overexposure to heat (Arauz,2000).

Effective biocontrol agents offer great potential to developalternative methods that are economical and suited for adop-

∗ Corresponding author. Tel.: +251 25 5530331; fax: +251 25 5530316.E-mail address: [email protected] (A. Ayalew).

0925-5214/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.postharvbio.2008.03.007

and bacteria isolated from unmanaged mango trees in different ecolo-s antagonistic towards Colletotrichum gloeosporioides, the cause of mangocteria, five yeasts and two filamentous fungi were evaluated in this study.trates of the isolates inhibited spore germination and hyphal growth of C.ates significantly reduced severity of anthracnose on artificially inoculatedinuta isolate B-62-13, Stenotrophomonas maltophilia L-16-12, a member of

dida membranifaciens F-58-22, and the yeast isolate B-65-23 which, basedsents an undescribed species, were effective on naturally infected fruit.y (lesion development) below 5% during much of the 12 d experimentalated fruit reached 29%. B. diminuta and the yeast B-65-23 were as effec-55 ◦C for 5 min. Further investigations on the mechanisms of biocontrololates, particularly the bacteria, for use on edible fruit are warranted. Onlytes showed a potential for the control of mango anthracnose on harvested

© 2008 Elsevier B.V. All rights reserved.

tion by the small-scale mango industry. However, few biocontrolagents have been reported against postharvest diseases of tropicalfruit. Among the limited isolates, a nonpathogenic strain of Col-

letotrichum magna (Prusky et al., 1993), isolate 558 of Pseudomonasfluorescens (Koomen and Jeffries, 1993), a strain of Bacillus sp. (DeJager et al., 2001), Bacillus licheniformis (Govender and Korsten,2006), and preharvest application of the yeast Rhodotorula minuta(Patinovera et al., 2005) have been found effective for the controlof postharvest diseases of mango.

This paper reports on the antagonism of microorganismsinitially isolated from mango towards C. gloeosporioides invitro and the potential of isolates of Brevundimonas diminuta,Stenotrophomonas maltophilia, an Enterobacteriaceae, Candida mem-branifaciens, and a possibly new yeast species for the control ofanthracnose development on harvested mango fruit.

2. Materials and methods

2.1. Isolates of antagonists and the pathogen

The potential biocontrol agents were selected from a screeningof 608, 563 and 402 isolates of bacteria, yeasts and filamentousfungi, respectively. They were initially isolated from mango leaves,blossoms and fruit sampled from unmanaged trees in different

Page 2: Postharvest biological control of anthracnose (Colletotrichum gloeosporioides) on mango (Mangifera indica)

Biolog

For tests on naturally infected fruit, antagonist propagule sus-

Y. Kefialew, A. Ayalew / Postharvest

ecological regions and habitats of Ethiopia. Sub-samples of leavesor blossoms (1 g each) were ground and suspended in 9 mL of ster-ile distilled water. Fruit peel cut into pieces (1 g) was shaken in 9 mLsterile distilled water for 10 min on a wrist-action shaker. Aliquotsof ten-fold dilutions of the suspension (leaves or blossoms) or wash(fruit) were used for isolation of bacteria and fungi by the pour platemethod. Nutrient agar (NA) was used for isolation of bacteria whilemalt extract agar (MEA) amended with 50 mg L−1 streptomycin sul-phate and 50 mg L−1 ampicillin was used for fungi. After incubationof NA plates for 2 d and MEA plates for 5–7 d at 25 ◦C, developingcolonies were characterized based on gross morphology and rep-resentative isolates were transferred to fresh NA (bacteria) or MEA(fungi) plates. Pure cultures grown on NA and MEA were maintainedat 4 ◦C until they were used. Effective isolates were selected basedon their inhibitory effect towards C. gloeosporioides in dual culturetests.

The antagonists used in this study comprised four bacterialisolates, namely B-62-13, B-66-15, L-16-12, and L-19-13; two fila-mentous fungi, F-47-35 and F-59-31; and the yeast isolates B-65-23,B-66-24, F-02-26, F-58-22, and L-69-21. The bacterial isolates werelater identified as B. diminuta (B-62-13), S. maltophilia (L-16-12),and as a member of Enterobacteriaceae (L-19-13). Among the yeasts,isolate F-58-22 was identified as C. membranifaciens while B-65-23appeared to be a new species. The remaining isolates of the studywere not identified. Identifications were made at the Global PlantClinic, CAB International, UK (Enquiry number 117/06); the bacteriawere identified based on fatty acid analysis and the yeasts throughinternal transcribed spacer (ITS) sequence analysis. All isolates havebeen deposited at the Plant Pathology Herbarium of Haramaya Uni-versity, Ethiopia.

An isolate of C. gloeosporioides from symptomatic mango fruitobtained at Bisidimo, eastern Ethiopia, grown on MEA was main-tained at 4 ◦C and used throughout the study.

2.2. In vitro evaluation of antagonistic activity

Each antagonist isolate was evaluated in two ways. First, cul-ture filtrates were tested for their effects on spore germinationand hyphal growth of C. gloeosporioides. Bacterial isolates weregrown in nutrient broth (NB) and yeasts or mycelial fungi inmalt extract broth (MEB); 250 mL of NB or MEB in 500 mL Erlen-meyer flasks were inoculated and then incubated on a shaker(120 rpm) at 25 ◦C. After a 3-d incubation, culture filtrates of bac-teria or yeast isolates were obtained by sedimenting the cellsthrough centrifugation at 6000 rpm (about 2500 g) for 10 min and

passing the supernatant through sterile Whatman-1 filter paper.Culture filtrates of filamentous fungi were similarly obtained butafter incubation for 15 d. Aliquots of 0.5 mL of undiluted culturefiltrate of each isolate were then pipetted into three replicatesingle-well depression slides and allowed to dry in a laminarflow cabinet. Meanwhile, a spore suspension of C. gloeosporioideswas prepared from a 10-d-old MEA culture. Conidia were trans-ferred with a sterile loop to 10 mL sterile distilled water containing0.05% Tween 80; the concentration of spores was determined usinga hemacytometer and adjusted with sterile distilled water, and0.5 mL aliquots (containing 100–150 spores) were then pipettedinto each depression slide. The slides were incubated in a humidchamber at 25 ◦C for 48 h. Data on spore germination and germtube or hyphal length were recorded every 12 h under a com-pound microscope. A conidium was considered to have germinatedwhen a germ tube at least half the length of the conidium wasobserved.

In the second in vitro test, the effects of cell (yeasts/bacteria)or spore suspensions of the isolates on spore germination andhyphal growth of C. gloeosporioides were evaluated. Bacterial and

y and Technology 50 (2008) 8–11 9

yeast cells were harvested from the broth cultures used in the pre-ceding test. Following centrifuging and decanting of the culturefiltrate, the cells were rinsed and resuspended in sterile distilledwater. The concentration of cells was determined with a hema-cytometer and adjusted to 1 × 105 cells mL−1 with sterile distilledwater. For isolates of filamentous fungi, both antagonists and thepathogen, spore suspensions were prepared by flooding 10-d-oldMEA cultures with sterile distilled water containing 0.05% Tween80 and suspending spores using sterile loop. The concentration ofspores was determined using a hemacytometer and adjusted to1 × 105 spores mL−1 with sterile distilled water. Cell or spore sus-pensions of the antagonists were pipetted (0.5 mL each) into threereplicate depression slides and 0.5 mL of the spore suspension ofC. gloeosporioides was then pipetted to each slide. The slides wereincubated, and data were collected as described for the culturefiltrate test.

2.3. In vivo tests

Antagonist isolates were tested for their effects on mangoanthracnose development on harvested fruit. Fruit of the mangocultivar ‘Amaba Kurfa’ were obtained from Bisidimo in Hararghe,Ethiopia, from two apparently healthy trees (for test with artificialinoculation) and two severely infected trees (for test under natu-ral infection). The trees were marked about 6 months prior to thetests and were kept free from any pesticide sprays. Mature fruit ofcomparable size and color class were used for the tests.

For test on artificially infected fruit, cell or spore sus-pensions of each antagonist isolate containing 103, 105, and107 propagules mL−1 of sterile distilled water were prepared, fol-lowing the respective procedures described under the secondin vitro test. Likewise, a spore suspension of C. gloeosporioidescontaining 105 conidia mL−1 was prepared. Mango fruit weresurface-sterilized by dipping in 1% sodium hypochlorite for 10 min,rinsed in sterile distilled water and inoculated by dipping in thespore suspension of C. gloeosporioides. After incubation in plasticbag for 15 h, fruit were dip-inoculated with the antagonist sus-pensions while control fruit were dipped in sterile distilled water.Each treatment was applied to five fruit (replicates) arranged incompletely randomized design (CRD). Following a further 12 d incu-bation (2 d within plastic bags and 10 d without), disease severitywas scored on a 1–5 scale as described by Koomen and Jeffries(1993), where 1 = no spots, 2 = 1–3 spots, 3 = 4–6 spots, 4 = 7–12spots 5 = >12 spots or 30% of the mango surface affected.

pensions of 105 or 107 cells or spores mL−1 were prepared asdescribed above. Within 24 h after harvest, mango fruit weretreated by dipping in the antagonist suspensions. Benchmarktreatment of dipping fruit in water at 55 ◦C for 5 min, as recom-mended by Dodd et al. (1997), and untreated controls in whichfruit were dipped in distilled water were included. The exper-iment was set up in CRD with five replications (five fruit foreach treatment) and repeated. Fruit were incubated for 12 d.After the first 48 h, anthracnose severity was recorded daily byvisual assessment of the percentage fruit area covered by lesionsaccording to the diagrammatic scale developed by Corkidi et al.(2006).

2.4. Statistical analysis

Analysis of variance (ANOVA) was carried out with the statisti-cal software SPSS for Microsoft Windows, v. 12 (SPSS Inc., Chicago,USA). Percentage data on spore germination and on disease sever-ity were arc-sine transformed before statistical analysis. Severityratings were subjected to square root transformation. Mean com-parisons were made using Dunnett t-test.

Page 3: Postharvest biological control of anthracnose (Colletotrichum gloeosporioides) on mango (Mangifera indica)

10 Y. Kefialew, A. Ayalew / Postharvest Biology and Technology 50 (2008) 8–11

Table 1ation

hal le

.5

.2

.3

.3

.3

.6

.9

.4

.4

.7

.3

.3 81.1 189.3

ated spores 48 h after treatment; NS indicates a value not significantly different from theunnett t-test).

reduced anthracnose severity levels below 5% for much of the 12 dexperimental period. Moreover, the fruit treated with these five iso-lates had only small inconspicuous spots that did not affect marketappeal and were not thus rendered unmarketable by anthracnoseinfections. On the other hand, infected fruit in the other treatmentsand the untreated control (with more than 20 and 29% terminalseverity, respectively) were generally non-marketable within 2 d ofdisease onset, i.e., 7–8 d after treatment. The five isolates showedeffectiveness, as a single postharvest treatment, to control fruitdecay due to anthracnose within acceptable standard. Accordingto Janisiewicz and Korsten (2002), the levels of decay acceptable inpostharvest systems are below 5%. Effects of the five isolates andthe hot water treatment on anthracnose severity were significant(P < 0.05) throughout the 12 d period.

Hot water treatment controlled mango anthracnose more effec-tively than the results presented by Arauz (2000). Fruit were dippedin hot water at 55 ◦C for 5 min in the present study as compared to

Table 2Effect of microbial antagonists on severity of anthracnose on artificially inoculatedmango fruit

Antagonist isolate Disease severity

Effect of culture filtrates and suspensions of microbial antagonists on spore germin

Antagonist isolate Culture filtrate

Spore germination (%) Hyp

BacteriaB. diminuta B-62-13 6.8 24B-66-15 (unidentified) 11.3 19S. maltophilia L-16-12 7.5 45Enterobacteriaceae L-19-13 7.0 37

Filamentous fungiF-47-35 (unidentified) 14.2 45F-59-31 (unidentified) 38.2 73

YeastsB-65-23 (unknown) 11.3 53B-66-24 (unidentified) 8.4 54F-02-26 (unidentified) 4.8 46C. membranifaciens F-58-22 10.2 66L-69-21 (unidentified) 10.5 77

Control 81.1 189

Values are means of three replications; hyphal growth was determined from germincontrol; all other values are significantly lower than that of the control at P < 0.05 (D

3. Results and discussion

3.1. In vitro antagonism towards C. gloeosporioides

Culture filtrates of all the antagonists significantly (P < 0.05)reduced germination of spores of C. gloeosporioides and averagegrowth of its hyphae from germinated spores (Table 1). The rateof germination of untreated (control) spores was about 81%. Thelowest level of germination (4.8%), with 94% reduction over the con-trol, was observed in spores treated with culture filtrate of the yeastF-02-26. Cell suspensions of all bacterial and yeast isolates signifi-cantly reduced conidial germination and average length of hyphaefrom germinated conidia of C. gloeosporioides. Spore suspensions ofboth the fungal isolates also significantly (P < 0.05) affected hyphalelongation as compared to the control (Table 1).

It should be acknowledged that culture broths were not sub-jected to ultra-filtration and the resultant filtrates used in thepresent study might have contained cells or spores of antagonists.In spite of that, comparisons between activities of the culture fil-trates and the suspensions indicated that inhibitory substanceswere involved. Postharvest biocontrol agents have been describedto work by inhibiting target pathogens through antibiosis, compe-

tition, production of lytic enzymes, parasitism, induced resistanceand competition (Janisiewicz and Korsten, 2002), but few organ-isms antagonize by a single mechanism (Atlas and Bartha, 1987).

3.2. Effect of antagonists on mango anthracnose development

All isolates of the antagonists significantly reduced anthrac-nose severity on fruit that had been artificially inoculated with C.gloeosporioides (Table 2). The antagonists generally kept the diseaserating below 2 (corresponding to one to three anthracnose spots).The treatments showed increased effect on disease severity withincrease in propagule concentrations. Earlier investigations alsodemonstrated direct relationship between the population densityof an antagonist and the efficacy of postharvest biological controltreatment (McLaughlin et al., 1990; Wilson et al., 1993; Hong et al.,1998). On the other hand, one of the ideal characteristics of an idealantagonist is to be effective at low concentration.

When tested on naturally infected fruit, the bacterial antago-nists B. diminuta isolate B-62-13, S. maltophilia L-16-12 and theEnterobacteriaceae isolate L-19-13 (Fig. 1A) as well as the yeast iso-lates B-65-23 (unknown) and F-58-22 (C. membranifaciens) (Fig. 1B)

and hyphal growth of C. gloeosporioides

Cell or spore suspension

ngth (�m) Spore germination (%) Hyphal length (�m)

15.6 49.614.2 33.110.9 29.913.1 36.3

74.6 NS 109.375.1 NS 145.6

16.4 162.119.4 53.35.7 145.625.0 74.720.9 47.5

Antagonist concentration (numberof cells or fungal spores mL−1)

Pooled mean

×103 ×105 ×107

Bacterial isolatesB. diminuta B-62-13 2.2** 1.6** 1.6** 1.8B-66-15 (unidentified) 2.2** 2.0** 1.8** 2.0S. maltophilia L-16-12 2.2** 1.8** 1.6** 1.9Enterobacteriaceae L-19-13 3.0* 2.0** 1.8** 2.3

Filamentous fungal isolatesF-47-35 (unidentified) 2.4** 2.0** 1.6** 2.0F-59-31 (unidentified) 3.2 NS 2.0** 2.0** 2.4

Yeast isolatesB-65-23 (unknown) 2.6** 2.2** 1.8** 2.2B-66-24 (unidentified) 2.8* 2.6** 1.4** 2.3F-02-26 (unidentified) 4.2 NS 4.0 NS 3.0* 3.7C. membranifaciens F-58-22 2.8* 1.6** 1.6** 2.0L-69-21 (unidentified) 2.0** 2.0** 1.8** 1.9

Control 5.0

Values are means of five replications; disease severity was measured on a 1–5 scaleon fruit inoculated with C. gloeosporioides spore suspension (105 spores mL−1) 15 hprior to antagonist treatment, and incubated for 12 d; NS indicates a value not sig-nificantly different from the control; all other values are significantly lower than thecontrol at. *P < 0.05, and **P < 0.01 (Dunnett t-test).

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Y. Kefialew, A. Ayalew / Postharvest Biolog

Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of fruit.Ann. Rev. Phytopathol. 40, 411–441.

Fig. 1. Anthracnose development on naturally infected mango fruit treated withbacterial (A) and yeast (B) antagonists at 105 to 107 cells mL−1 (A: B-62-13 = B.diminuta, L-16-12 = S. maltophilia, and L-19-13 = member of Enterobacteriaceae; B:F-58-22 = C. membranifaciens, and B-65-23 possibly represents an undescribedspecies; all other isolates are unidentified; hot water = 55 ◦C for 5 min; con-trol = sterile distilled water).

53 ◦C for 3 min in that report. In general, postharvest dips of fruitin hot water are considered as moderately effective against mangoanthracnose, particularly under high disease pressure, unless theyare applied in combination with fungicides (Arauz, 2000). Theeffective heat treatment is often near the level injurious to thecommodity, so both temperature and duration must be preciselycontrolled (Barkai-Golan and Phillips, 1991; Arauz, 2000). In prac-tice, hot water treatment is applied on a large scale where facilitiesare available, but is unsuited for the small-scale fruit industry.

Testing isolates on artificially inoculated fruit allowed evalua-tion of their in vivo activity under uniform disease pressure, sincenatural infection could be irregular or minimal. However, therewas no direct relation between the relative effectiveness of theantagonists on artificially inoculated fruit and naturally infectedfruit (Table 2 and Fig. 1). In the former case, antagonists wereapplied 15 h after inoculation of fruit with C. gloeosporioides. Thisduration was reported to be sufficient to allow germination of

conidia of C. gloeosporioides (Koomen and Jeffries, 1993). In nature,postharvest anthracnose of mango originates from infections ofdeveloping fruit in the orchard that remain quiescent, possibly asdormant appressoria or subcuticular hyphae, until ripening of thefruit (Arauz, 2000).

Some of the potential biocontrol agents identified in the presentstudy have been reported to have roles in the biological con-trol of plant diseases. These include B. diminuta against Fusariumculmorum and Rhizoctonia solani (De Boer et al., 2007), S. mal-tophilia against Verticillium dahliae (Berg et al., 1994) and sugarbeet damping-off disease (Nakayama et al., 1999), and C. mem-branifaciens against Penicillium expansum (Valdebenito-Sanhuezaand Cattanio, 2003). In pack house trials on biocontrol of mangoanthracnose, Govender and Korsten (2006) reported the presenceof several species of Enterobacteriaceae in association with mango.Earlier, a species of Enterobacteriaceae was reported as effectiveagainst Botrytis cinerea (Guinebretiere et al., 2000).

B. diminuta B-62-13 and the yeast isolate B-65-23 exhibited dis-ease control effects which were statistically at par with the hotwater treatment indicating their potential for developing alter-native methods. B-65-23 appeared to represent an undescribed

y and Technology 50 (2008) 8–11 11

species; “this organism could not be identified by ITS sequenceanalysis. Top match was 94% to a sequence assigned to Candidarugosa but this does not represent sufficient identity for a species(or even genus) level identification” (Global Plant Clinic, CAB Inter-national, UK, Identification Report). Further studies to establish theidentity of isolate B65-23 would be required. Moreover, as indi-cated by Fravel (1988), understanding the mechanism of biocontrolis imperative to achieve practical utilization of antagonists.

Acknowledgments

We thank the Global Plant Clinic and particularly Paula Kellyand Lisa Offord (CABI Bioscience, UK), for kind help on identifica-tion of microbial isolates. The financial and material support fromGambella Agricultural Research Center and Ethiopian AgriculturalResearch Organization is gratefully acknowledged.

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