Biocontrol Agent Of Postharvest Diseases Research Articles

Yeasts and Bacterial Consortia from Kefir Grains Are Effective Biocontrol Agents of Postharvest Diseases of Fruits.

Fungal pathogens in fruits and vegetables cause significant losses during handling, transportation, and storage. Biological control with microbial antagonists replacing the use of chemical fungicides is a major approach in postharvest disease control, and several products based on single antagonists have been developed but have limitations related to reduced and inconsistent performance under commercial conditions. One possible approach to enhance the biocontrol efficacy is to broaden the spectrum of the antagonistic action by employing compatible microbial consortia. Here, we explore commercial kefir grains, a natural probiotic microbial consortium, by culture-dependent and metagenomic approaches and observed a rich diversity of co-existing yeasts and bacterial population. We report effective inhibition of the postharvest pathogen Penicillium expansum on apple by using the grains in its fresh commercial and milk-activated forms. We observed few candidate bacteria and yeasts from the kefir grains that grew together over successive enrichment cycles, and these mixed fermentation cultures showed enhanced biocontrol activities as compared to the fresh commercial or milk-activated grains. We also report several individual species of bacteria and yeasts with biocontrol activities against Penicillium rots on apple and grapefruit. These species with antagonistic properties could be further exploited to develop a synthetic consortium to achieve enhanced antagonistic effects against a wide range of postharvest pathogens.

Genome Sequence, Assembly and Characterization of Two Metschnikowia fructicola Strains Used as Biocontrol Agents of Postharvest Diseases.

The yeast Metschnikowia fructicola was reported as an efficient biological control agent of postharvest diseases of fruits and vegetables, and it is the bases of the commercial formulated product “Shemer.” Several mechanisms of action by which M. fructicola inhibits postharvest pathogens were suggested including iron-binding compounds, induction of defense signaling genes, production of fungal cell wall degrading enzymes and relatively high amounts of superoxide anions. We assembled the whole genome sequence of two strains of M. fructicola using PacBio and Illumina shotgun sequencing technologies. Using the PacBio, a high-quality draft genome consisting of 93 contigs, with an estimated genome size of approximately 26 Mb, was obtained. Comparative analysis of M. fructicola proteins with the other three available closely related genomes revealed a shared core of homologous proteins coded by 5,776 genes. Comparing the genomes of the two M. fructicola strains using a SNP calling approach resulted in the identification of 564,302 homologous SNPs with 2,004 predicted high impact mutations. The size of the genome is exceptionally high when compared with those of available closely related organisms, and the high rate of homology among M. fructicola genes points toward a recent whole-genome duplication event as the cause of this large genome. Based on the assembled genome, sequences were annotated with a gene description and gene ontology (GO term) and clustered in functional groups. Analysis of CAZymes family genes revealed 1,145 putative genes, and transcriptomic analysis of CAZyme expression levels in M. fructicola during its interaction with either grapefruit peel tissue or Penicillium digitatum revealed a high level of CAZyme gene expression when the yeast was placed in wounded fruit tissue.

Unraveling the Tritrophic interactions between fruit host-pathogen-antagonist in the post-harvest environment

Fruits and vegetables post-harvest losses due to the attack of several fungal pathogens can reach levels of up to 50% depending on the commodity. Besides the economic losses, some of these fungi may represent a potential health risk because of mycotoxin production. To control post-harvest diseases of fruits, synthetic fungicides, when permitted, are the primary means used. However, the emergence of fungicide-resistant strains and the willingness to use safer and eco- friendly treatments, have generated interest in the development of alternative non-chemical methods to reduce post-harvest losses. Biological control using microbial antagonists has emerged as one of the most promising alternatives to fungicides, either alone or as part of an integrated pest management. The antagonistic activity of biocontrol agents has been widely demonstrated, however the mechanisms of action of most biocontrol agents of post-harvest diseases has not yet been fully understood which is important to know for a successful implementation of post-harvest biocontrol technology. Therefore a part of my PhD focused on this aspect; we have successfully cloned, expressed in P. pastoris and confirmed the involve- ment of a novel endochitinase gene MfChi and the protease gene ALP5 in the biocontrol activity of the antagonistic yeasts Metschnikowia fructicola AP47 and Aureobasidium pullulans PL5 successively. Our work proved that the alkaline serine protease ALP5 and the endochitinase MfChi could be developed as post-harvest treatments with antimicrobial activity for fruits undergoing a short shelf life to control Botrytis cinerea, Monilinia fructicola and Monilinia laxa on pome and stone fruits. Moreover, besides developing biocontrol agents as alternative approaches to chemicals to control the pathogens, we should also be familiar with the decaying agents, such as their nature and the molecular basis of the infection. Due to recent technological advances, sequencing has revolutionized biological research and has become the forefront of bio- logical experimentation in the last decade. Therefore, we have sequenced for the first time Penicillium griseofulvum PG3 which is associated with blue mould decay, the most important post-harvest disease of pome fruit worldwide. The fungal pathogen has the ability to simultaneously produce both detrimental and beneficial Secondary Metabolites (SM). Our work aimed to analyse some important SM clusters present in the studied strain in order to gain insight into SM synthesis in P. griseofulvum. Genome-wide analysis of PG3 genes revealed a complete putative gene cluster for patulin biosynthesis, and partial griseofulvin and roquefortine C clusters. Besides the bioinformatics analysis of these gene clusters, we quantified the SM production in vitro and during disease development on apple. Furthermore, the SM detected in infected apple were examined by studying the expression kinetics of their key genes under controlled conditions. In addition, we found additional SM clusters in PG3, including those potentially responsible for the synthesis of penicillin and cyclopiazonic acid. These findings provide relevant information to understand the molecular basis of SM biosynthesis in P. griseofulvum and this resource will allow further research directed to the overexpression or blocking of specific SM synthesis, to assess its potential in terms of biotechnological applications for beneficial SM, such as griseofulvin.

The ability of a cold-adapted Rhodotorula mucilaginosa strain from Tibet to control blue mold in pear fruit.

Cold-adapted yeasts were isolated from soil samples collected in Tibet and evaluated as potential biocontrol agents against blue mold (Penicillium expansum) of pear fruit in cold storage. YC1, an isolate identified as Rhodotorula mucilaginosa, was found to exhibit the greatest biocontrol activity among the different isolates that were screened. A washed cell suspension of YC1 exhibited the best biocontrol activity among three different preparations that were used in the current study. A concentration of 10(8) cells/ml reduced the incidence of decay to 35%, compared to the control where decay incidence was 100%. A higher intracellular level of trehalose and a higher proportion of polyunsaturated acids present in YC1, was associated with increased the tolerance of this strain to low temperatures, relative to the other strains that were evaluated. The increased tolerance to low temperature allowed the YC1 strain of yeast to more effectively compete for nutrients and space in wounded pear fruit that had been inoculated with spores of P. expansum and placed in cold storage. The present study demonstrated the ability to select cold-adapted yeasts from cold climates and use them as biocontrol agents of postharvest diseases of fruit placed in cold storage.

English

  Aureobasidium pullulans, popularly known as black yeast, is one of the most widespread saprophyte fungus associated with wide range of terrestrial and aquatic habitats, in temperate and tropical environment. It is a polymorphic fungus that is able to grow in single yeast-like cells or as septate, polykaryotic hyphae showing synchronous conditions, with budding cells. This fungus has been exploited potentially for commercial production of various enzymes (amylase, xylanase, pectinase, etc). Single cell protein, alkaloids and polysaccharide, especially pullulan, an exopolysaccharide, is a linear α-d-glucan connected with α-1,4 glycosidic bond mainly of maltotriose repeating units interconnected by α-1,6 linkages. Pullulan has been considered as one of the important polysaccharide for production of biodegradable plastics. More than 300 patents for applications have been developed. It is the only fungus which produce higher amount of pullulan and has been exploited all over the world. The fungus has excellent genetic make-up to produce various important metabolites at commercial production with limited species. Some of the A. pullulans have potential antagonistic activity against a number of phytopathogenic fungi used as bio-control agents of post-harvest diseases. It has been found to be tolerant to many metal ions which are common pollutants of soil and water. Several strains of this fungus have ability to degrade xenobiotic compounds. In the light of the above facts, this review article has emphasized on the orientation, morphology, biochemical characteristics, habitats and its economic potentials with special reference to pullulan.    Key words: Aureobasidium pullulans, single cell protein, xenobiotic compounds, pullulan.

Development and application of a SCAR marker to monitor and quantify populations of the postharvest biocontrol agent Pantoea agglomerans CPA-2

Pantoea agglomerans CPA-2 is an effective biocontrol agent of postharvest diseases of citrus and pome fruit. A monitoring technique was developed for its identification and to quantify its populations. The methodology used consisted of (i) searching for a semi-selective medium, (ii) identification of molecular markers and (iii) monitoring population dynamics in a commercial trial. As a semi-selective medium, Malonate Broth Agar supplemented with tetracycline hydroxychloride and incubation at high temperature (max. of 40 °C) facilitated the selective recovery of P. agglomerans CPA-2 colonies. The RAPD technique was applied to a collection of 13 strains of P. agglomerans, including CPA-2. Among the 12 primers tested, OPL-11 amplified a fragment (about 720 bp) specific to strain CPA-2. On the basis of this fragment, two SCAR markers were amplified using a primer pair derived from OPL-11 elongation. A first SCAR marker of 720 bp was specifically amplified for the strain CPA-2 and a second one of 270 bp was obtained for all P. agglomerans strains tested, including CPA-2. Commercial trials demonstrated a significant reduction of decay with the treatment of formulated cells of P. agglomerans CPA-2. Population dynamics of CPA-2 in commercial trials were determined on fruit surfaces and in the environment using both the classical plating technique and PCR with SCAR primers. In general, no significant differences were observed between results obtained from the two methods. On fruit surfaces, 1 day after CPA-2 applied its population by classical methods was 4.37 × 10 6 cfu wound −1 and at the end of the experiment the population increased to 5.8 × 10 5 cfu wound −1. The percentages of colonies identified as P. agglomerans CPA-2 at these sampling times using SCAR primers were 90 and 95%, respectively. Population dynamics in the environment to evaluate the environmental fate of P. agglomerans CPA-2 showed that it has a limited persistence and limited capacity for dispersion.

IMPROVING QUALITY AND SAFETY OF FRESH FRUITS AND VEGETABLES AFTER HARVEST BY THE USE OF BIOCONTROL AGENTS AND NATURAL MATERIALS

World trends are moving towards reduced pesticide use in fresh fruit and vegetables. Along with this trend, several physical and biological means have been evaluated as safer alternatives for the use of chemical fungicides. The use of microbial antagonists for the control of postharvest diseases received special attention, and has been extensively investigated. Most of the reported yeast and bacteria antagonists were naturally occurring on fruit surfaces. Microbial biocontrol agents of postharvest diseases have been criticized mainly for not providing as consistent or broad-spectrum control as synthetic fungicides. The first generation of biological controls for postharvest spoilage relied on the use of single antagonists. Perhaps it is unrealistic for us to expect disease control comparable to synthetic fungicides by the use of single antagonists. It can be expected that enhancing efficacy of biocontrol agents of postharvest diseases to an acceptable level will utilize a combination of different biological and physical means. As we learn more about the fundamental basis underlying the protective effect of microbial antagonists, bioactive compounds, and induced resistance, more effective methods of formulating, applying and combining complementary biological approaches for additive and/or synergistic effects will emerge. So far the results obtained with the different combinations of biological chemical and physical means demonstrate the potential of this multifaceted approach as a viable alternative to synthetic fungicides.

Control of postharvest rots of sweet cherries and table grapes with endophytic isolates of Aureobasidium pullulans

Abstract Fifty-one endophytic isolates of Aureobasidium pullulans were obtained from the flesh of sweet cherries and extensively screened to evaluate their biocontrol activity against postharvest rots of sweet cherries and table grapes. Preliminary analysis of all isolates by randomly amplified polymorphic DNA (RAPD) with three different primers showed the presence of a high genetic variability and enabled isolates not showing any genetic difference to be discarded. Thirty-five isolates with different RAPD electrophoresis patterns had a wide range of biocontrol activity against Botrytis cinerea and Monilinia laxa on single-wounded berries of sweet cherries and table grapes with a reduction of decay from 10 to 100%. Two isolates (533 and 547) significantly reduced B. cinerea on table grape berries also when applied 6, 12, and 24 h after the pathogen inoculation. In a 2-year period of investigation (1998–1999), a reduction of total rots ranging from 32 to 80% (sweet cherries) and from 59 to 64% (table grape) was achieved with isolates 533 and 547 applied after harvest. Preharvest applications of isolate 547 significantly reduced postharvest rots of sweet cherries and table grapes by 47 and 38%, respectively. On the whole, isolates 533 and 547 were more effective than A. pullulans L47, a biocontrol agent of postharvest diseases with a known activity. Population studies demonstrated that isolate 547 was able to survive under field conditions, to increase its population during cold storage, and to penetrate the flesh of sweet cherries when applied during flowering.

Molecular Detection of Strain L47 of Aureobasidium pullulans, a Biocontrol Agent of Postharvest Diseases.

The strain L47 of Aureobasidium pullulans is an effective biocontrol agent of postharvest diseases. When applied in the field before harvesting it requires a specific monitoring method to evaluate colonization and dispersal in the environment. The randomly amplified polymorphic DNA technique (RAPD) was used for a preliminary screening of A. pullulans genetic variability among 205 isolates. This approach allowed the selection of a 1.3-kb fragment (L4) present solely in isolates L47 and 633. In Southern blots, a digoxigenin (DIG)-labeled L4 amplicon specifically recognized the corresponding fragment present in the polymorphic pattern of L47 and 633. The L4 fragment was cloned, sequenced, and used to design two sequence-characterized amplification region (SCAR) primers and a 242-bp riboprobe. Both the SCAR primers and the 242-bp DIG-labeled riboprobe were highly specific for L47. In classical polymerase chain reaction (PCR), with a series of 10-fold dilutions of L47 DNA, the limit of detection was 20 pg/μl. The Ap13 primer was also modified to obtain a Scorpion primer for detecting a 150-bp amplicon by fluorescence emitted from a fluorophore through a self-probing PCR assay. This assay specifically recognized the target sequence of L47 strain over a number of other A. pullulans isolates in field-treated grape berry washings. The limit of detection was 105 cells per ml, i.e. 10 times higher than the limit of the CFU method. The method is also proposed as a way to demonstrate the ability of L47 strain to penetrate the epidermis of sweet cherry fruits and to track it in the mesocarp.

Pilot tests of Candida sake (CPA-1) applications to control postharvest blue mold on apple fruit

Abstract The yeast Candida sake (strain CPA-1) was tested as a biocontrol agent of postharvest diseases, primarily blue mold, caused by Penicillium expansum on apple fruits. In a semi-commercial trial with non-injured fruits stored in air at 1°C, a concentration of 1.6×10 6 colony forming units/ml (CFU/ml) of C. sake reduced the incidence of decayed fruits by more than 70%. Over a period of three seasons, in commercial trials the efficacy of CPA-1 applied in a drench was evaluated and compared with the fungicides imazalil and thiabendazole+folpet. The application of C. sake at 10 7 CFU/ml resulted in a reduction in the incidence of decay to a level equal to that with imazalil (375 ppm) and higher than that with thiabendazole (425 ppm)+folpet (1000 ppm). Population of the biocontrol agent increased on the surface of wounded fruits 5-fold and decreased on the surface of non-wounded fruits more than 10-fold in the first 60 days in storage at 1°C. The viability of C. sake was not reduced after 30 min immersion in benomyl, sulfur, flusilazol, ziram, thiabendazole or diphenylamine. Conversely, captan, imazalil, and ethoxyquin decreased C. sake viability and would not be compatible with it used at commercial rates. The yeast was able to grow in culture at temperatures from 1 to 34°C.

Biological Control of Postharvest Diseases of Apple and Pear under Semi-commercial and Commercial Conditions Using Three Saprophytic Yeasts

The yeastsCryptococcus laurentii(strain HRA5),Cryptococcus infirmominiatus(strain YY6), andRhodotorula glutinis(strain HRB6) were tested as biocontrol agents of postharvest diseases of apple and pear in semi-commercial and commercial trials. The yeasts effectively controlled decay when applied in a drench or line spray. The yeasts were not adversely affected when treated fruits were stored in a controlled atmosphere consisting of 1% oxygen and 99% nitrogen. In a commercial trial, the most effective treatments for control of blue mold of pear were a combination ofC. laurentiiandC. infirmo-miniatus(91% control) and the commercially recommended high rate (528 μg/ml) of thiabendazole (88% control). In the commercial apple trial, the most effective treatments for blue mold wereC. infirmo-miniatuscombined with 264 μg/ml thiabendazole (91% control),C. infirmo-miniatuscombined withC. laurentii(84% control), and thiabendazole alone at 528 μg/ml (79% control). The combination ofC. laurentiiwith 264 μg/ml of thiabendazole was significantly more effective for control of blue mold on pear than thiabendazole at 528 μg/ml whenever any thiabendazole-resistant spores were present in the inoculum.