Revista Mexicana de Fitopatología

ISSN: 0185-3309

mrlegarreta@prodigy.net.mx

Sociedad Mexicana de Fitopatología, A.C.

México

Luna Romero, Isaac Juan; Carvajal Moreno, Magda; Flores Martínez, Arturo; Ferrera Cerrato, Ronald

Possibility of Biological Control of Aspergillus flavus with Pseudomonas fluorescens on Maize Ear

Revista Mexicana de Fitopatología, vol. 18, núm. 1, enero-junio, 2000, pp. 50- 54

Sociedad Mexicana de Fitopatología, A.C.

Texcoco, México

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50/ Volumen 18, Número 1, 2000

Possibility of Biological Control of Aspergillus flavus withPseudomonas fluorescens on Maize Ear

Isaac Juan Luna-Romero, Depto. de Microbiología, Escuela Nacional de CienciasBiológicas-Instituto Politécnico Nacional (ENCB-IPN), Prol. Carpio y Plan de Ayala, Col.Sto. Tomás, México D.F. CP 11340; Magda Carvajal-Moreno, Depto. de Botánica,Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria,Apdo. Postal 70-233, México, D.F. CP 04510; Arturo Flores-Martínez, ENCB-IPN; yRonald Ferrera-Cerrato, Centro de Edafología, Colegio de Postgraduados, Km 35.5Carret. México-Texcoco, Montecillo, Edo. de México CP 56230. Correspondence to:magdac@servidor.unam.mx

(Received: March 29, 2000 Accepted: August 23, 2000)

Abstract. Pseudomonas fluorescens isolates with confirmedantagonistic activity against Aspergillus flavus, were testedto determine their capacity to inhibit fungal infection on maizeear grains at the milk stage. The best method to obtain a100% infection was through the induction of a wound in thegrain, before inoculating the fungus. There was a reductionof infection produced by A. flavus from 35.8% to 3.2% ofmaize ear bioassays, in direct proportion to the bacterialconcentration. Inoculation of A. flavus of maize ear silks inthe outdoor glashouse, produced infection similar to thenatural one. Bacterial antagonistic isolates provided goodprotection against A. flavus. Isolate Co-47 gave the bestprotection to silks and grains, followed by isolates Tez-226and Tez-118. Isolate Co-81 was less efficient on in vitro tests,but in the field there was a good infection inhibition by thisisolate. Isolate Ba-72 was the least effective in controllingfungal infection. Maize grain infection by A. flavus in thefield was higher than in the outdoor glasshouse even withthe antagonic bacterial treatments and the fungus.

Additional keywords: Aflatoxins, antagonistic bacteria,biocontrol, fungal infection.

Resumen. Se probaron aislamientos de Pseudomonasfluorescens con actividad antagónica confirmada contraAspergillus flavus, para determinar su capacidad de inhibir lainfección causada por este hongo en granos de mazorca demaíz en estado lechoso. El mejor método para obtener 100%de infección fue provocar una herida al grano antes deinocular al hongo. Hubo una reducción de 35.8% a 3.2% de lainfección de A. flavus en bioensayos en mazorca, directamenteproporcional a la concentración bacteriana. La inoculaciónde A. flavus en estigmas de mazorcas de maíz en invernaderoa cielo abierto, produjo una infección similar a la natural; losaislamientos antagónicos bacterianos protegieron bien contraA. flavus. El aislamiento Co-47 dió la mejor protección a

estigmas y granos, seguido por los aislamientos Tez-226 yTez-118. El aislamiento Co-81 fue menos eficiente en pruebasin vitro, pero inhibió bien la infección en granos. El aislamientoBa-72 fue el menos efectivo para controlar la infección fúngica.La infección de grano de maíz por A. flavus al campo fuemayor que la del invernadero a cielo abierto, aún con lostratamientos con bacterias antagónicas y el hongo.

Palabras clave adicionales: Aflatoxinas, bacteriasantagónicas, biocontrol, infección fúngica.

The presence of antagonistic microorganisms in nature is akey factor for the control of crop diseases (Mukerji and Garg,1988). Bacterial populations with antagonistic capacityagainst plant pathogens are variable, being determined byenvironmental factors such as weather, soil and host plants(Burpee, 1990). There are many beneficial bacteria in agronomicsystems that have not been studied (Weller, 1988). Somebacteria have antagonistic ability to inhibit plant pathogensby production of antibiotics and other compounds such asbacteriocins, acids (Kerr, 1980; Weller and Cook, 1983) orsiderophores (Leong, 1986; Neilands, 1981). There are plant-growth rhizobacteria, they help to develop plants throughsuppression of damaging bacterial populations (Kloepper etal., 1980; Schroth and Hancock, 1982). The use of beneficialmicroorganisms will promote healthy crops with goodproduction for growers. Microoganisms that biologicallycontrol plant pathogens usually do not work at the samelevel as in vitro, and only a few such as Trichoderma, Bacillusand Pseudomonas (Cook, 1991) have been applied to thefield. Aspergillus flavus damages maize and other cereals(Hesseltine, 1976) and produces aflatoxins that are mutagenic,teratogenic and carcinogenic metabolites. These aflatoxinsaffect all living creatures that consume contaminated foods(Allcroft, 1969; Shank, 1977). Chemical products and resistantvarieties are used to control this fungus (Pettit et al., 1971;

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Scott and Zummo, 1987; Widstrom, 1987); but until now, therehas been little work using antagonistic bacteria for biologicalcontrol (Cuero et al., 1991). This paper describes theantagonistic capacity of different isolates of Pseudomonasfluorescens as a biological control option in maize to controlA. flavus infection in an outdoor glasshouse and in the field.

MATERIALS AND METHODSThe antagonistic isolates of Pseudomonas fluorescens usedin this research, were isolated from a previous study, wherethey were selected out of 3425 bacterial isolates fromrhizosphere and phylloplane, and their inhibiting capacityagainst A. flavus was previously confirmed (Luna, 2000).These isolates were named as Co-47, Co-81, Tez-118, Tez-226,Ba-72 and Ba-252, depending on the places where they weresampled (Co-Cocotitlan, State of Mexico; Tez = Tezoyucan,State of Mexico, and Ba = Bajío State of Guanajuato).Infectivity tests. A preliminary test was performed to establishthe best inoculation method to obtain a 100% infection by A.flavus of maize ears with grains at the milk stage. Two differentmethods were used to inoculate maize ears: a) 100 grains,from ears separated from the plant, were inoculated placing adrop of a conidial suspension (3.5 x 106 conidia/ml) on eachgrain; b) A wound was made with a dissection needle in thegrain tip, placing the conidial suspension, at the aboveconcentration. This experiment was repeated 3 times. Controlswere maize ears inoculated with distilled sterile water inwounded or healthy grains. Treated ears were incubated inmoist chambers at 28°C for 8 days.Bioassays in grains at the milk stage. The method ofinoculation with a wound was applied to 100 maize grains atthe milk stage. Three bacterial concentrations (3 x 107, 3 x 108

and 3 x 109 bacteria/ml) in accordance to the Mac Farlandscale (Kiraly et al., 1974) were used. The bacterial suspensiondrop was left to dry for 20 minutes, and immediately 0.01 ml ofA. flavus conidial suspension (3.5 x 106 /ml) was placed in thesame place. The experiment was repeated 5 times. Inoculatedears were incubated in a moist chamber, in 30 x 20 x 7.5 cmplastic boxes, at 28°C. As negative controls, maize grainswere inoculated with distilled sterilized water. Other controlswere maize grains inoculated with antagonistic bacterialisolates with the purpose to find their innocuous ordeletereous effect. As a positive control, 100 maize grainswere inoculated only with A. flavus (3.5 x 106) to produceinfection. The effects of the bacterial isolate and suspensionconcentration on infected grains was analysed by BifactorialDeviance Analysis, using a binomial error type distributionand a logit linkage function. This procedure is similar tovariance analysis, but it is used for proportion data thatfrequently do not fulfill the demands for a common varianceanalysis (Crawley, 1993; Mc Gullagh and Nelder, 1983).Antagonistic effect in outdoor glasshouse and in the field.Outdoor glasshouse tests. Inhibition trials were conductedin the glasshouse with the bacteria during the summer of1997 in Mexico City, to evaluate their effect on A. flavus of

maize. The following experimental design was performed: Eightrows were sown in an area of 4 x 4 m, with a between rowspacing of 50 cm. For each line, six seeds in groups of sixwere sown with a distance of 40 cm between groups. Onceplants reached 50 cm height, they were thinned to eliminatethe less vigorous and yellow plants, leaving three plants persite. Inoculation of bacterial isolates and of A. flavus werecompleted in accordance to the methodology proposed byJones et al. (1980) who reproduced maize grain infection byspraying silks with a conidial suspension of A. flavus (3.5 x106). Five treatments were established, the inoculation ofantagonistic bacteria plus A. flavus and two controltreatments, one positive with A. flavus and the other withsterile distilled water only. For each treatment 15 plants wereinoculated in their exposed silks. When the silks had abrownish colour, 15 days after flowering, they were sprayedwith 2 ml of a bacterial suspension (3 x 109) and 25 min later, 2ml of fungal conidial suspension was applied. Immediatelyafter being sprayed, each ear was labelled and covered with aplastic and a paper bag for 5 days; afterwards, the bags wereremoved to allow the ear to develop normally. The ears wereharvested at physiological maturity, when the grain hadreached the black layer stage and had 30% seed moisture(Zeleny, 1977). Ears were transported to the Plant Pathologylaboratory (National School of Biological Sciences, IPN,Mexico City), and dried at 36°C, until they reached a 10-12%grain moisture. Five ears from each treatment were randomlyselected and shelled, and 100 grains were selected todetermine percentage infection by A. flavus. To promote fungaldevelopment, one hundred grains were treated with sodiumhypochloride at 2% for 5 min, and later placed in Petri disheswith PDA (Armentrout, 1988). For each bacterial isolate, therewere five replications of 100 grains each. Plates wereincubated at 28°C for 10 days, and percentage infectiondetermined by fungal growth from grains sown in PDA.Field condition tests. To determine the antagonistic capacityof the bacterial isolates, field trials were conducted in a plotnear Cocotitlan, State of Mexico, with mild dry weather andsummer rains. Maize was sown in rows with a distance of 50cm between plants and 50 cm between rows. The maize wasmanaged with recommended agronomic practices (fertilizer,insecticides and weeding). Treatments were the same as thosein the outdoor glasshouse tests; five bacterial isolates plusthe fungus A. flavus and two controls, positive with thefungus and negative only with sterile distilled water. Fifteenplants, with complete formed ears were randomly selected foreach treatment. Bacteria and fungus inoculations were carriedout when silks were completely exposed and had a brownishcolour, 15 days after flowering. Bacteria and fungusconcentrations were the same as in the outdoor glasshousetests. Similarly, bacteria were first inoculated and later thefungus. Then, ears were covered for five days with plasticand paper bags, to give proper moisture conditions tomaintain the activity of the inoculated microorganisms. Oncethe ears had reached physiological maturity, 30% grain

Revista Mexicana de FITOPATOLOGIA/53

differ significantly from the treatment with only A. flavus(Table 2). Antagonistic behaviour of the different isolates inoutdoor glasshouse conditions and field did not differsignificantly (X2 = 5.01, P= 0.4049), the infection field rateswere around three times higher than in the outdoor glasshousein all treatments.

DISCUSSIONWounds in maize grains were important to obtain A. flavusinfection, during all their development from ear grainsformation until their physiological maturity. Wounds facilitatefungal penetration and infection of maize tissues, therefore,the importance of insects, animal injury, sowing andharvesting activities to spread fungal damage in the crop.The control programmes must be directed to avoid woundsinflicted by insects, animal injuries and agronomical cropactivities, and protect the ear during its development andharvest. The fungus was not able to penetrate the graindirectly; in this study, there were optimal conditions ofhumidity and temperature, however, the fungus did not infect.Antagonistic bacteria inoculated in wounded grains couldbe considered innocuous because they did not damage thetissues of maize grains, they only produced darkening of

tissues, similar to that produced with distilled water (controls).This might be due to the oxidation of phenolic compounds incontact with air (Hammerschmidt, 1985). Innocuity was alsosupported by previous experiments, such as those carriedout on tobacco and potato soft rot, that determine thephytopathogenic or saprophytic character of bacteria (Luna,2000). Making wounds to the grains before inoculation withA. flavus, allowed to evaluate the capacity of each isolate toinhibit A. flavus development in maize grains at the milk stageof development. High concentrations of antagonistic bacteria(3 x109) might protect the grain against fungal infection betterthan low concentrations (3x 107). Variability of antagonisticbehaviour of the tested isolates showed the differentantagonistic potential of each isolate. Although isolatesbelong to the same taxonomic group of Pseudomonasfluorescens, their intraspecific genetic characteristics providesignificant differences in their capacity to produce differentantibiotics. The antagonistic potential of isolate Co-47 toinhibit A. flavus infection was better than the remainderisolates, supporting the results obtained in vitro. Isolate Ba-252 lost its antagonistic capacity against A. flavus bycontinuous transfers, it is a very unstable isolate and is notrecommended for biological control. Co-47, Co-81, Ba-72. Tez-118 and Tez-226, however, maintained their antagonisticactivity with the same number of transfers and the sameamount of handling as with Ba-252. There was a low infectionrate in outdoor glasshouse tests in relation to the field assaysfor almost all the treatments, although sometimes there werenot significant differences due to environmental conditions.Native isolates Tez-118 and Tez-226, had more advantagesover foreign isolates or those introduced from other regions,because they are more adapted to temperature and humidityconditions of Tezoyuca. The number of infected grainsobtained in the outdoor glasshouse and in the field, afterspraying maize silks with A. flavus conidia, agrees with Joneset al. (1980), and shows the capacity of A. flavus to colonizeand naturally infect maize through silks. Percentage infectionof controls, i.e. when the fungus was inoculated alone, werelow in outdoor glasshouse and in the field. However, thisinfection would be enough to cause serious problems instorage, if the seed was not properly dried and temperaturesare warm leading to the production of aflatoxins. When silkswere protected with antagonistic bacteria, percentageinfection was reduced especially with isolates Co-47 and Co-81, therefore they might be considered the most promising toproduce a biological system of control against A. flavus,especially in regions where they are native or where there aresimilar environmental conditions.

AckowledgementsTo the Institute of Biology, Directing Office of Support toAcademic Personnel (DGAPA) for the PhD scholarship,National Autonomous University of Mexico (UNAM) and tothe National Polytech Institute.

Table 1. Percentage infection by Aspergillus flavus in maizeears previously inoculated by antagonistic bacteria.Bacterial isolates Infection by A. flavus in Maize Ears

Bacterial Concentration 107 108 109

Co-47 + Af 35.8 (12.7) 26.8 ( 6.7) 3.2 (2.3)Co-81 + Af 54.8 (22.1) 29.4 (12.0) 15.6 (6.1)Tez-118 + Af 54.4 (14.8) 50.2 (5.9) 4.2 (2.1)Tez-226 + Af 69.8 (14.4) 33.4 (14.8) 3.4 (2.1)Ba-72 + Af 61.4 (17.3) 30.4 (12.4) 13.8 (6.1)Ba-252 + Af 97.7 ( 4.4) 88.0 (6.8) 82.0 (20.8)Control Af 100Values in parenthesis indicate standard deviation.Control Af = Aspergillus flavus inoculated at 3.5 x 106 conidia/ml.

Table 2. Protecting effect of antagonistic bacteria on infectionpercentage of Aspergillus flavus in maize ears.

Af = Aspergillus flavus.

Treatments Infection of Maize Ears (%) Glasshouse Standard Field Standard error errorA. flavus control 8.8 0.73 24.4 0.81Co-47 + Af 1.0 0.55 8.4 1.17Co-81 + Af 1.4 0.51 6.4 0.93Tez-118 + Af 3.4 0.51 16.6 0.93Tez-226 + Af 4.6 0.68 18.4 0.81Ba-72 + Af 7.6 0.51 25.0 1.1

54/ Volumen 18, Número 1, 2000

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