Postharvest Biology and Technology 38 (2005) 57–65

Semi-commercial evaluation ofBacillus licheniformistocontrol mango postharvest diseases in South Africa

Veloshinie Govendera, Lise Korstena,∗, Dharini Sivakumara,b

a Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria 0002, South Africab Postharvest Technology Group, Agro and Food Technology Division, Industrial Technology

Institute, 363 Baudhaloka Mawatha, Colombo 7, Sri Lanka

Received 17 October 2004; accepted 7 April 2005

Abstract

Efficacy of the biocontrol agentBacillus licheniformiswas evaluated under semi-commercial conditions on a mango packingline to control anthracnose and stem-end rot on the mango cultivar ‘Keitt’. Mango fruit were treated with either the biocontrolagent applied in hot water (45◦C) followed by a quarter strength prochloraz dip or with the biocontrol agent applied on itsown in hot water. These treatments were compared to the untreated control and commercially used prochloraz hot water dip.Treated fruit were dried and waxed on the commercial packing line. Fruit subjected to the prochloraz-biocontrol hot watercombination showed reduced anthracnose and stem-end rot incidence after market simulated conditions of low temperaturestorage at 10◦C with 90% RH and at room tempeature (20◦C at 75% RH for 7 days). This integrated treatment retained thefruit colour and firmness with high marketability most effectively, compared to the other treatments. The biocontrol agent wase r ana higheri red fungalp©

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ffectively recovered from treated fruit after 21 days storage at 10◦C, 90% RH (6 log units) and declined to 3 log units aftedditional 7 days storage at 20◦C, 75% RH. Total recovered bacterial and yeast populations on the fruit surface were

n fruit subjected to the integrated treatment and held under both storage conditions. In contrast, the total recoveopulation was higher on untreated control fruit.2005 Elsevier B.V. All rights reserved.

eywords:Anthracnose; Biocontrol agent; Hot water treatment; Prochloraz; Stem-end rot

. Introduction

South African Mango (Mangifera indica L.) ishe fifth most important subtropical crop with annnual production of 24,584 tonnes (De Jager, 1999).

∗ Corresponding author. Tel.: +27 12 420 3295;ax: +27 12 420 4588.E-mail address:lkorsten@fabi.up.ac.za (L. Korsten).

Postharvest diseases that cause considerable losthe mango industry are anthracnose caused byCol-letotrichum gloeosporioides(Penzig) and stem-enrot caused byBotryosphaeriaspp. Managementthese diseases relies primarily on the applicatiocopper-based preharvest sprays (Lonsdale and Kotze,1993; Ploetz and Prakash, 1997) and postharvest hwater dips incorporating fungicides (Johnson et al1997; Swart et al., 2002). These practices have prov

925-5214/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.postharvbio.2005.04.005

58 V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65

relatively effective in protecting the crop against pre-and postharvest pathogen infection and in extendingthe storage life of mango fruit during overseas ship-ment. Susceptibility of mango fruit to postharvest dis-eases increases after harvest and prolonged storage asa result of physiological changes in the fruit, favouringpathogen development (Eckert et al., 1996).

The adverse effects of synthetic chemical residueson human health (Lichtenberg and Zilberman, 1987)and the environment (Weaver et al., 1990) have leadto intensified world-wide research efforts to developalternative control strategies. In addition, availabilityof fungicides to be used on export fruit has been effec-tively reduced particularly for smaller niche crops suchas mango. In light of the above, there is increased sci-entific interest in biological control of plant pathogensas an alternative form of disease control (Cooke, 1993).

Biological control using microbial antagonists hasemerged as one of the most promising alternatives, usedeither on its own or as part of an integrated control strat-egy to reduce the use of synthetic fungicides (Fan andTian, 2001; El Ghaouth et al., 2002). In South Africa,biological control programmes on mango were initi-ated in 1987, resulting in the successful evaluation ofBacillus licheniformisas a biocontrol agent (Burgerand Korsten, 1988). It effectively reduced the incidenceof anthracnose as a pre- or postharvest application(Korsten et al., 1991). Preharvest applications providedmore consistent control compared to the postharvestapproach (Korsten et al., 1992). The efficiency of theb n itw at al dK tet io-cs har-v nd tor u-l

2

2

rolp outh

Africa) was mixed with water (400 l) to obtain a finalliquid concentration of 107 cells ml−1 according to themanufacturer’s instructions.

2.2. Fruit and storage conditions

Freshly harvested mango fruit cv. Keitt was obtainedfrom Bavaria Estate in the Mpumulanga Province,South Africa. Each postharvest treatment consistedof 9 replications of 10 fruit. Fruit were harvested atcommercial maturity and were subjected to posthar-vest treatments under semi-commercial conditions on amechanised packing line at Bavaria Estates. These andother experiments and treatment combinations werecarried out over three growing seasons, during whichtime mango fruit from different cultivars were evalu-ated. The results obtained showed similar trends andhave been reported previously (Govender, 2004). Datapresented in this report present only the final semi-commercial trial using ‘Keitt’ fruit.

2.3. Postharvest treatments undersemi-commercial conditions

A set of fruit was separately subjected to the fol-lowing three postharvest treatments in the commercialpacking line:

(1) Integrated treatment: A hot water 5 min dip(400 l water) at 45◦C incorporating the prepared

on

1-outhof

cialt-ruit.

(th

rated

(at

(

iocontrol agent could further be improved wheas applied with the recommended fungicide, used

ower concentration (Korsten et al., 1992; Silimela anorsten, 2001). The aim of this study was to evalua

he efficacy of a commercial formulation of the bontrol product Mangogreen (B. licheniformis) underemi-commercial conditions to control mango postest diseases (anthracnose and stem-end rot) aetain overall marketability of the fruit during simated export conditions.

. Materials and methods

.1. Commercial biocontrol product

Commercial powder formulation of the biocontroduct Mangogreen (Stimuplant cc., Pretoria, S

liquid biocontrol product at a final concentratiof 107 cfu ml−1 (B. licheniformis), followedby a 20 s dip in a prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy) ethyl] imidazole-carboxamide) (Saarchem, Johannesburg, SAfrica) bath prepared at a quarter strengththe recommended rate, followed by commerdrying at 35◦C and waxing with Citrashine (Cirashine PVT Ltd., Johannesburg) 1 l per tonne f

2) Chemical treatment: Hot water dip at 45◦C for5 min, followed by 20 s dipping in a water bacontaining prochloraz at the recommend(1.8%), followed by commercial drying anspraying with wax.

3) Biocontrol treatment: Hot water dip at 45◦Cfor 5 min, incorporating the biocontrol agent107 cfu ml−1 (B. licheniformis).

4) Control: Untreated fruit served as control.

V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65 59

At completion of the treatments, fruit were commer-cially packed (12 fruit per box) and stored for 21 daysat simulated sea export conditions (10◦C, 90% RH) inthe commercial cold room at Bavaria Estate. After coldstorage, fruit were held at marketing conditions (20◦Cfor 7 days) to simulate ripening on the retail shelf.

2.4. Fruit ripening and postharvest diseasedevelopment during storage

Incidence of anthracnose or stem-end rot wasrecorded as the ratio of fruit showing disease symp-toms against the total number of fruit treated (Pruskyet al., 1999). Severity of the above mentioned posthar-vest diseases was determined according to a 1–5 scale(1: no symptoms; 2: 1/4 of the fruit surface show-ing symptoms; 3: 1/2; 4: 3/4; 5: entire fruit surfacedecayed) (Wszelaki and Mitchem, 2003). Fruit mar-ketability was assessed after 7 days storage at 20◦Caccording to a scale rating (1–2: very poor; 3–4:poor; 5–6: fair; 7–8: good; 9–10: excellent) (Sivakumaret al., 2002). Colour development was assessed visu-ally and the colour index was expressed using a scalefrom 1 (green) to 5 (full colour). Fruit firmness wasassessed for ten fruit from each treatment, on oppos-ing sides of each fruit by a hand held firmness tester(Bareiss Prufgeratebau GmbH, DKD, Germany).

2.5. Recovery of the commercial biocontrol agentand postharvest pathogens during storage

at-m ge at2 er’ss ath( an-n asfi -b ofw iona mlo entw Bio-l malte4 ry io-c ns

and total surface bacterial, yeast and fungal popu-lations were expressed as colony forming units permilliter (cfu ml−1) (De Jager, 1999). The antagonistcolonies were distinguished from background florabased on colony morphology and confirmation of iden-tity of selected representative isolates. Identity ofB.licheniformiswas confirmed using the API diagnos-tic kits (Logan and Berkeley, 1981). The backgroundmicroflora was obtained from control fruit.

2.6. Statistical analysis

Data were subjected to analysis of variance(ANOVA) using theGenStat for Windows (2000)sta-tistical package (Release 4.2, fifth ed., VSN Interna-tional, Oxford). Fisher’s protectedt-test, least signifi-cant difference (LSD) at the 1% level of significance,was performed.

3. Results

3.1. Postharvest treatments and disease incidence

Anthracnose and stem-end rot were not observedafter low temperature storage; however, the develop-ment of these two diseases were noted during simulatedmarketing conditions. Under commercial conditions,the integrated treatment was the most effective andreduced both anthracnose and stem-end rot severitya .F ctivet eritya bio-c ntly( bothd inte-g

3

e e tos otht olourd io-ch st

Six fruit were randomly selected from each treent after low temperature storage and after stora0◦C, and washed in 500 ml quarter strength Ringolution (Merck, Johannesburg) in an ultrasonic bUltrasonic Manufacturing Company (Pty) Ltd., Johesburg) at 25◦C for 30 s. The surface washing wltered through a 0.22�m filter in a vacuum assemly. The filters were cut aseptically in quarters,hich one was transferred to 9 ml Ringer’s solutnd subjected to a serial dilution. Aliquots of 0.1f each dilution from each postharvest dip treatmas transferred to potato dextrose agar (PDA) (

ab, Johannesburg), Standard-1 agar (Biolab) andxtract agar (Biolab) plates and incubated at 32◦C,8 h for growth of bacteria and 28◦C for 4–5 days foeast and fungi. Observation on survival of the bontrol agent (B. licheniformis), postharvest pathoge

nd incidence significantly (P< 0.001) (Fig. 1a and b)urther, the integrated treatment was more effe

han the chemical treatment in reducing the sevnd incidence of both diseases. Although theontrol agent incorporated in hot water significaP< 0.001) reduced the incidence and severity ofiseases, it was not as effective as the chemical orrated treatments.

.2. Fruit ripening and marketability

Colour development was significantly (P< 0.001)nhanced after transferring fruit from cold storagimulated marketing conditions (data not shown). Bhe integrated and chemical treatments retarded cevelopment while the fruit from the control and bontrol treatment showed a significantly (P< 0.001)igher colour index (Fig. 2a). However, the highe

60 V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65

Fig. 1. Effect of postharvest treatments on (a) anthracnose and stem-end rot incidence and (b) severity on mango after storage at 10◦C, 90%RH for 21 days and 20◦C, 75% RH for 7 days. Integrated treatment: hot water;B. licheniformis; prochloraz; wax. Chemical treatment: hotwater; prochloraz; wax. Biological treatment:B. licheniformis; hot water. Postharvest disease severity was rated according to 1–5 scale (1: nosymptom; 2: 1/4 of the fruit surface showing symptoms; 3: 1/2; 4: 3/4; 5: entire fruit). Within bars, means followed by the same letter do notdiffer significantly at the 1% level of the Fisher’s Protected Least Significant Test.

Fig. 2. Effect of postharvest treatments on (a) colour, marketability and (b) firmness of mango after storage at 10◦C, 90% RH for 21 days and20◦C, 75% for 7 days. Integrated treatment: hot water;B. licheniformis; prochloraz; wax. Chemical treatment: hot water; prochloraz; wax.Biological treatment:B. licheniformis; hot water. Within bars, means followed by the same letter do not differ significantly at the 1% level ofthe Fisher’s Protected Least Significant Test.

V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65 61

Fig. 3. Appearance of fruit treated with integrated postharvest treatments held at 12◦C, 90% RH for 21 days and 20◦C, 75% RH for 7 days.Integrated treatment: hot water;B. licheniformis; prochloraz; wax. Chemical treatment: hot water; prochloraz; wax. Biological treatment:B.licheniformis; hot water.

percentage marketable fruit was obtained from theintegrated treatment. Although the chemical treatmenthad revealed more marketable fruit compared to thecontrol and biological treatment, it was significantly(P< 0.001) less than the integrated treatment.

Fruit firmness was observed to decline significantly(P< 0.001) after transfer from cold storage to roomtemperature (data not shown). Fruit dipped in the bio-control or chemical treatments and control fruit hadsignificantly (P< 0.001) lower firmness than the fruitfrom the integrated treatment after 7 days at 20◦C(Figs.2band3).

3.3. Recovery of the biocontrol agent postharvestpathogens and total microbial population

Recovery of the biocontrol agent (B. licheniformis)was significantly (P< 0.001) higher in fruit subjected

to the integrated treatment compared to the bio-logical treatment at both storage temperatures. Therecovery ofB. licheniformisfrom fruit subjected tothe integrated treatment was 6 log units after coldstorage for 21 days, and it declined to 4 log unitsafter 7 days at 20◦C (Fig. 4a and b). The recov-ery of the postharvest pathogens (C. gloeosporioides,Botryosphaeriaspp. andB. theobromae) was notedto increase when fruit were transferred from 10 to20◦C. However, fruit subjected to the integrated treat-ment had significantly (P< 0.001) lower recovery ratesof postharvest pathogens, compared to other treat-ments. Recovery of postharvest pathogens increasedmore than 4 log units in fruit subjected to biolog-ical treatment and in untreated fruit at 20◦C after7 days.

The total microbial population on the fruit surfacewas observed to increase when fruit were transferred

62 V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65

Fig. 4. Effect of integrated postharvest treatments on recovery ofB. licheniformisand postharvest pathogens from mango after storage at (a) 10◦C,90% RH for 21 days and (b) 20◦C, 75% for 7 days. Integrated treatment: hot water;B. licheniformis; prochloraz; wax. Chemical treatment:hot water; prochloraz; wax. Biological treatment:B. licheniformis; hot water. Postharvest pathogens:C. gloeosporioides, B. theobromae,Botryosphaeriaspp. Within bars, means followed by the same letter do not differ significantly at the 1% level of the Fisher’s Protected LeastSignificant Test.

from 10 to 20◦C (Fig. 5a and b). The total micro-bial population was significantly (P< 0.001) higher infruit subjected to the integrated treatment, compared toother treatments. The recovery of yeast populations washigher and fungal populations significantly (P< 0.001)lower on fruit subjected to the integrated treatment,compared to the other treatments at both storage tem-peratures. The chemical treatment did not have a sig-nificant effect on the yeast population when fruit weretransferred from 10 to 20◦C. However, the recoveryof fungal populations from chemical treated fruit wasobserved to increase up to 3 log units.

The recovery of yeast populations was higher onfruit subjected to the biological control treatment after10◦C. Further, the fungal populations also increasedto 6 log units when these fruit were held at 20◦C. Thehighest recovery of fungal and the lowest yeast popu-lations were found on the surface of untreated controlfruit at both storage temperatures. The total yeast pop-ulation increased during the transfer of fruit from 10to 20◦C in all fruit, irrespective of the treatments. Theyeast population was significantly (P< 0.001) lower in

untreated fruit at 10◦C, for 21 days, which increasedto 3 log units at 20◦C after 7 days under marketingconditions. Fruit treated with the biocontrol treatmentrevealed similar high populations of yeast at 20◦C.However, an increase in yeast population was noted infruit subjected to the integrated treatment during trans-fer from 10 to 20◦C.

4. Discussion

The integrated treatment effectively controlledpostharvest diseases and retained marketability of thefruit most effectively. The potential ofBacillusspp. asbiological control agents has been reported previously(Huang et al., 1992; Korsten et al., 2000; Batta, 2004).The biocontrol agentB. licheniformis, water-basedformulation effectively controlled postharvest diseaseswhen applied as a preharvest spray (Korsten et al.,1992). The findings of this trial proved that thecommercially formulated product ofB. licheniformisMangogreen could effectively control anthracnose

V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65 63

Fig. 5. Effect of integrated postharvest treatments on total microbial population of mango fruit surfaces after storage at (a) 10◦C, 90% RHfor 21 days and (b) 20◦C, 75% for 7 days. Integrated treatment: hot water;B. licheniformis; prochloraz; wax. Chemical treatment: hot water;prochloraz; wax. Biological treatment:B. licheniformis; hot water. Within bars, means followed by the same letter do not differ significantly atthe 1% level of the Fisher’s Protected Least Significant Test.

and stem-end rot under semi-commercial conditions.This is the first report of a commercial product ofB.licheniformis evaluated in an integrated postharvestsemi-commercial application with quarter strengthprochloraz on mango fruit. Similar observationswere reported on the control of postharvest citrusdiseases with combinations ofCryptococcus infirmo-miniatus using low concentrations of thiabendazole(Chand-Goyal and Spotts, 1996) and in control ofBotrytis storage rot on strawberries and table grapeswith Aureobasidium pullulansin combination withlow concentrations of fungicides (Ippolito et al.,1997).

Addition of B. licheniformis to reduced rates ofthe chemical treatment improved the efficiency ofanthracnose and stem-end rot control in mangoesunder semi-commercial export conditions. Previousstudies indicated that the dip treatment of mangoesin aqueous solutions ofB. licheniformisreduced bothdiseases up to 25% (Govender and Korsten, 2001). Theefficiency ofB. licheniformiswas improved by using

quarter strength prochloraz applied in a wax treatment(Korsten and Lonsdale, 1993; Govender, 2004). Thesuccess of commercialisation of a biocontrol agentsdepends on the potential of practical use, its abilityto colonise on fruit surface in storage and to persist(Wisniewski and Wilson, 1992). Within this contextthe biocontrol agent,B. licheniformissurvived the hotwater temperature of 45◦C and the low temperaturestorage for a 21 days period. The viability of theB.licheniformisat 45◦C was high and could be explainedby the heat-shock effect caused by the temperaturedifference similar to that reported bySingh andDaverall (1984)andGovender (2004). The heat-shockeffect resulted in a transformation from a dormant endospore stage to the viable active stage (Govender, 2004).Therefore, product performance was not adverselyaffected by temperature variations. The survival ofB.licheniformis in waxed fruit is evident from its highrecovery from the integrated treatment which wassimilar to that reported byGamagae et al. (2004). Waxcoating influenced the survival ofB. licheniformis

64 V. Govender et al. / Postharvest Biology and Technology 38 (2005) 57–65

on the fruit surface and increased the total microbialpopulation due to the modified atmosphere syner-gism. A similar observation was reported bySpottset al. (1998)with sweet cherries andCryptococcusinfirmo-miniatusin a modified atmosphere package.The populations ofB. licheniformisremained at stablelevels on the surface of mango fruit under storage andmarketing simulation conditions; therefore, it couldbe suggested thatB. licheniformiscould be applied inwax coatings for mangoes, as a protectant.

The high total microbial populations on fruit sur-faces subjected to the integrated treatment were similarto the densities reported byDe Jager (1999)on matureuntreated mango fruit. However, postharvest chem-ical treatments had a negative effect on the mangomicroflora. It is also evident in this study that theaddition of the biocontrol agent in the integrated treat-ment contributed to maintaining the natural microbialecological balance as reported byIppolito and Nigro(2000) and Gerhardson (2002). Also the integratedtreatment revealed a synergistic effect of prochloraz,B. licheniformis, wax and hot water on reducing thetotal fungal population and maintaining high bacterialyeast population on the fruit surface. It is known thathot water treatment hastens the ripening and degreen-ing process (Liu, 1978; Klein and Lurie, 1992) Hotwater treatment applied without waxing affected thenatural wax layer on the fruit surface, which enhancedfruit shriveling and senescence. This contributed to thehigher severity values found with the anthracnose ands herc tioni ar-k her ayect ity,c orth ofl df udyd d bO lr lPl ic,a ics

5. Conclusion

The mode of action ofB. licheniformiswas reportedas antibiosis due to the production of bioactive com-pounds and volatiles (Govender, 2004). It is importantto know the biocontrol agent–host–pathogen inter-actions before product registration (Janisiewicz andKorsten, 2002a,b). IncorporatingB. licheniformisinthe quarter strength concentration of prochloraz on thecommercial packing line provides a practical solutionto ensure ultimate fruit quality on the export market.

Acknowledgements

This work was partly supported by grants fromthe South African Mango Growers’ Association andTechnology and Human Resources for Industry Pro-gramme (THRIP), a partnership programme funded bythe Department of Trade and Industry and managed bythe National Research Foundation (NRF). We expressour thanks to Bavaria Estates, South Africa, for provid-ing their pack house to conduct this trial. The editorialassistance of Ms. C.A.M. Zeeman is greatly acknowl-edged.

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