Biocontrol Capacity Research Articles

EBR1 genomic expansion and its role in virulence of Fusarium species

Genome sequencing of Fusarium oxysporum revealed that pathogenic forms of this fungus harbour supernumerary chromosomes with a wide variety of genes, many of which likely encode traits required for pathogenicity or niche specialization. Specific transcription factor gene families are expanded on these chromosomes including the EBR1 family (Enhanced Branching). The significance of the EBR1 family expansion on supernumerary chromosomes and whether EBR1 paralogues are functional is currently unknown. EBR1 is found as a single copy in F.graminearum and other fungi but as multiple paralogues in pathogenic F.oxysporum strains. These paralogues exhibit sequence and copy number variation among different host-specific strains and even between more closely related strains. Relative expression of the EBR1 paralogues depends on growth conditions and on the presence of the single EBR1 gene in the core genome. Deletion of EBR1 in the core genome in different F.oxysporum strains resulted in impaired growth, reduced pathogenicity and slightly reduced biocontrol capacities. To identify genes regulated by EBR1, the transcriptomes of wild-type and Δebr1 strains were compared for both F.oxysporum and F.graminearum. These studies showed that in both species, EBR1 regulates genes involved in general metabolism as well as virulence.

English

Fusarium oxysporum is an abundant plant pathogen in the soil and is found in the rhizosphere of many plant species. The fungus has numerous formae speciales (f.sp.) that infect and cause wilt diseases of a variety of host plants. F. oxysporum f.sp lycopersici (Fol) and F. oxysporum f.sp lini (Foln) are known to infect tomato and flax, respectively. Fluorescent pseudomonads can suppress various soilborne diseases, and their efficacy has been related both to their antagonistic activities and to their rhizosphere competitiveness. In this study, the biocontrol capacity of plant growth-promoting rhizobacteria (PGPR) strains of Pseudomonas fluorescens against fusarium wilt of tomato and flax was evaluated both in vitro and in planta. The P. fluorescens strains exhibited substantial antagonistic activities against the two fungal isolates on three culture media tested: King B (KB), potato dextrose agar (PDA) and a mixture of both (KB + PDA). As compared to the untreated control, the mycelial growth decreased by 8.33 to 49.33%. Conidial germination and germ tube elongation were significantly inhibited and reduced, respectively, by 6.15 to 47.33% and by 1.63 to 45.45%. In bioassay trials, P. fluorescens strains significantly reduced the disease incidence and severity by 4.86 to 74.49% on tomato plants and 3.93 to 79.19% on flax plants. The different efficacy of the biocontrol agents could be due to the influence of several factors, including the efficiency of the strain, the type of pathogen and the host. Key words: Plant growth-promoting rhizobacteria (PGPR), Pseudomonas fluorescens, fusarium wilt, tomato, flax, biocontrol.

In vitro and in vivo antimicrobial efficacy of essential oils and individual compounds against Phytophthora parasitica var. nicotianae

To evaluate the antimicrobial effects of essential oils (EOs) from cassia, basil, geranium, lemongrass, cumin and thyme, as well as their major components, against Phytophthora parasitica var. nicotianae; to investigate morphological changes in hyphae and sporangia in response to treatment with cinnamaldehyde; and to further evaluate potential biocontrol capacities against tobacco black shank under greenhouse conditions. The results revealed that the extent of mycelial growth inhibition was primarily dependent on the composition and concentration of the EOs and the structure of individual compounds. Cinnamaldehyde had a significantly higher inhibitory effect on mycelial growth, formation of sporangia, and production and germination of zoospores in P. parasitica var. nicotianae in vitro, achieving complete inhibition of these phenotypes at 72, 36, 36 and 18 mg l(-1), respectively. Scanning electron microscopic observations revealed that cinnamaldehyde can cause considerable morphological degenerations of hyphae and sporangia such as cytoplasmic coagulation, shrivelled mycelia and sporangia aggregates and swelling and lysis of mycelia and sporangia walls. In vivo assays with cinnamaldehyde demonstrated that this compound afforded protective effect against tobacco black shank under greenhouse conditions in susceptible tobacco plants. The results of in vitro and in vivo bioassays, together with SEM imaging of the microstructure of P. parasitica var. nicotianae supported the possibility of using cinnamaldehyde as a potent natural biofungicide in the greenhouse. This study provides a theoretical basis for the potential use of cinnamaldehyde as commercial agents or lead compounds that can be exploited as commercial biofungicides in the protection of tobacco plants from P. parasitica var. nicotianae infection.

Efficacy and putative mode of action of native and commercial antagonistic yeasts against postharvest pathogens of pear

Putative mechanisms of action associated with the biocontrol capacity of four yeast strains (Cryptoccocus albidus NPCC 1248, Pichia membranifaciens NPCC 1250, Cryptoccocus victoriae NPCC 1263 and NPCC 1259) against Penicillium expansum and Botrytis cinerea were studied by means of in vitro and in situ assays. C. albidus(YP), a commercial yeast was also evaluated for comparative purposes. The yeast strains exhibited a variety of different mechanisms including: wound colonization, germination inhibition, biofilm formation, secretion of killer toxins, competition for nutrient and secretion of hydrolytic enzymes (protease, chitinase and glucanase). The relationship between strains (and their associated antagonist mechanisms) and in situ antagonist activity was also evaluated. Results indicate that mechanisms such as production of hydrolytic enzymes, the ability for colonization of wounds, production of killer toxin and inhibition of germination are the most important for biocontrol activity. Our study indicate that multiple modes of action may explain why P. membranifaciens NPCC 1250 and C. victoriae NPCC 1263 provided excellent control of postharvest pears disease.

Differential induction of grapevine resistance and defense reactions against Botrytis cinerea by bacterial mixtures in vineyards

Previous experiments showed that seven bacteria identified as Acinetobacter lwoffii (PTA-113 and PTA-152), Bacillus subtilis (PTA-271), Pantoea agglomerans (PTA-AF1 and PTA-AF2) and Pseudomonas fluorescens (PTA-268 and PTA-CT2) induced systemic resistance in grapevine against Botrytis cinerea. Based on these findings, we investigated biocontrol capacity of different mixtures under vineyard conditions over three consecutive years. Treatments with bacterial mixtures were shown to induce systemic resistance against B. cinerea on year 2. Efficacy and duration of such a disease control seemed to be reinforced on year three without renewal of bacterial treatments. Accordingly, the effectiveness of induced resistance varied with mixture type of bacteria and was accompanied by a stimulation of chitinase and β-1,3 glucanase activities in both leaves and berries. Interestingly, treatments with mixtures containing both selected strains of P. agglomerans (PTA-AF1 + PTA-AF2) appeared as the most effective in triggering systemically the plant defense reactions and reducing the symptoms of grey mould disease.

Burkholderia cepacia XXVI siderophore with biocontrol capacity against Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is the causal agent of anthracnose in mango. Burkholderia cepacia XXVI, isolated from mango rhizosphere and identified by 16S rDNA sequencing as a member of B. cepacia complex, was more effective than 6 other mango rhizosphere bacteria in inhibiting the model mango pathogen, C. gloeosporioides ATCC MYA 456. Biocontrol of this pathogen was demonstrated on Petri-dishes containing PDA by > 90 % reduction of surface colonization. The nature of the biocontrol metabolite(s) was characterized via a variety of tests. The inhibition was almost exclusively due to production of agar-diffusible, not volatile, metabolite(s). The diffusible metabolite(s) underwent thermal degradation at 70 and 121 °C (1 atm). Tests for indole acetic acid production and lytic enzyme activities (cellulase, glucanase and chitinase) by B. cepacia XXVI were negative, indicating that these metabolites were not involved in the biocontrol effect. Based on halo formation and growth inhibition of the pathogen on the diagnostic medium, CAS-agar, as well as colorimetric tests we surmised that strain XXVI produced a hydroxamate siderophore involved in the biocontrol effect observed. The minimal inhibitory concentration test showed that 0.64 μg ml(-1) of siderophore (Deferoxamine mesylate salt-equivalent) was sufficient to achieve 91.1 % inhibition of the pathogen growth on Petri-dishes containing PDA. The biocontrol capacity against C. gloeosporioides ATCC MYA 456 correlated directly with the siderophore production by B. cepacia XXVI: the highest concentration of siderophore production in PDB on day 7, 1.7 μg ml(-1) (Deferoxamine mesylate salt-equivalent), promoted a pathogen growth inhibition of 94.9 %. The growth of 5 additional strains of C. gloeosporioides (isolated from mango "Ataulfo" orchards located in the municipality of Chahuites, State of Oaxaca in Mexico) was also inhibited when confronted with B. cepacia XXVI. Results indicate that B. cepacia XXVI or its siderophore have the potential to be used as a biological control agent against C. gloeosporioides; thus diminishing environmental problems caused by the current practices to control this disease.

Biological Control of Rhizoctonia Root Rot on Bean by Phenazine- and Cyclic Lipopeptide-Producing Pseudomonas CMR12a

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.

Biocontrol of Potato White Mold Using Coniothyrium minitans and Resistance of Potato Cultivars to Sclerotinia sclerotiorum

This study was conducted in Bahar and Lalehjin, Hamadan, Iran to assess the biocontrol efficacy of Coniothyrium minitans Campbell against potato white mold caused by Sclerotinia sclerotiorum (Lib.) de Bary under field and greenhouse conditions. In addition, the resistance of common potato cultivars against S. sclerotiorum was determined in a greenhouse experiment. After straw inoculation of six potato cultivars (Pashandi, Istambouli, Agria, Marfauna, Alpha and Spartaan) with S. sclerotiorum, the least disease severity was observed in Spartaan and Marfauna. Agria showed the most susceptibility to S. sclerotiorum. Compared with the healthy control, different concentrations of C. minitans conidia (<TEX>$10^7$</TEX>, <TEX>$10^8$</TEX> and <TEX>$10^9$</TEX> conidia/mL) reduced disease severity under greenhouse condition, and a concentration <TEX>$10^9$</TEX> was the most effective treatment. During 2008 and 2009, four field trials were conducted to evaluate the efficacy of C. minitans in different soil and aerial applications on disease incidence of potato white mold. In 2008, soil application of <TEX>$Contans^{(R)}$</TEX> WG (a commercial product of C. minitans) showed the greatest biocontrol capacity whereas soil application of solid-substrate C. minitans was found inferior when compared with other treatments in both Bahar and Lalehjin field sites. In 2009, benomyl application was the most effective treatment in reducing disease incidence in both tested field sites.

Characterization of Bacillus isolates of potato rhizosphere from andean soils of Peru and their potential PGPR characteristics

Bacillus spp. are well known rhizosphere residents of many crops and usually show plant growth promoting (PGP) activities that include biocontrol capacity against some phytopatogenic fungi. Potato crops in the Andean Highlands of Peru face many nutritional and phytophatogenic problems that have a significant impact on production. In this context is important to investigate the natural presence of these microorganisms in the potato rhizosphere and propose a selective screening to find promising PGP strains. In this study, sixty three Bacillus strains isolated from the rhizosphere of native potato varieties growing in the Andean highlands of Peru were screened for in vitro antagonism against Rhizoctonia solani and Fusarium solani. A high prevalence (68%) of antagonists against R. solani was found. Ninety one percent of those strains also inhibited the growth of F. solani. The antagonistic strains were also tested for other plant growth promotion activities. Eighty one percent produced some level of the auxin indole-3-acetic acid, and 58% solubilized tricalcium phosphate. Phylogenetic analysis revealed that the majority of the strains belonged to the B. amyloliquefaciens species, while strains Bac17M11, Bac20M1 and Bac20M2 may correspond to a putative new Bacillus species. The results suggested that the rhizosphere of native potatoes growing in their natural habitat in the Andes is a rich source of Bacillus fungal antagonists, which have a potential to be used in the future as PGP inoculants to improve potato crop.

Microbiological control of soil‐borne phytopathogenic fungi with special emphasis on wilt‐inducingFusarium oxysporum

Plant diseases induced by soil-borne plant pathogens are among the most difficult to control. In the absence of effective chemical control methods, there is renewed interest in biological control based on application of populations of antagonistic micro-organisms. In addition to Pseudomonas spp. and Trichoderma spp., which are the two most widely studied groups of biological control agents, the protective strains of Fusarium oxysporum represent an original model. These protective strains of F. oxysporum can be used to control wilt induced by pathogenic strains of the same species. Exploring the mechanisms involved in the protective capability of these strains is not only necessary for their development as commercial biocontrol agents but raises many basic questions related to the determinism of pathogenicity versus biocontrol capacity in the F. oxysporum species complex. In this paper, current knowledge regarding the interaction between the plant and the protective strains is reviewed in comparison with interactions between the plant and pathogenic strains. The success of biological control depends not only on plant-microbial interactions but also on the ecological fitness of the biological control agents.

Use of two PGPR strains in the integrated management of blast disease in rice ( Oryza sativa) in Southern Spain

Biocontrol capacity of two plant growth-promoting rhizobacteria (PGPR) strains, against blast disease in rice paddy fields in Southern Spain was studied in three cropping seasons. Both strains ( Pseudomonas fluorescens Aur 6 and Chryseobacterium balustinum Aur 9) had already shown biocontrol capacity against pathogens, ability to induce systemic resistance against leaf pathogens and against salt stress in different plant species. Bacterial treatments were carried out on seeds and/or on leaves. Strains were inoculated individually and in combination. Protection against natural disease incidence was evaluated, and rice production and quality measured in 2005 and 2006 trials. In 2004, natural disease incidence was low (between 0.1% and 0.35% of damaged leaf surface) due to environmental conditions; under these conditions, both strains significantly protected plants against rice blast. In 2005, disease incidence was higher than in 2004, reaching higher values of affected leaf surface in controls. In these conditions, each strain individually protected rice against rice blast, although the combination of both strains was the most effective treatment. All three treatments (Aur 6, Aur 9 and Aur 6 + Aur 9) reached 50% protection in panicles, with Aur 9 being the most effective. In 2006, the most effective treatment was the combination of both strains on leaves in three physiological stages, suggesting a biocontrol mediated protection. On the other hand, when bacteria were applied to seeds, disease incidence decreased up to 50%, suggesting induction of systemic resistance. Finally, a direct relation between protection mediated by the PGPR and the increase in rice productivity (mT/ha) and quality (weight of 1000 seeds and number of intact grains after milling) was found.

Biocontrol capacity of wild and mutant Trichoderma harzianum (Rifai) strains on Rhizoctonia solani 618: effect of temperature and soil type during storage

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.12 No.4, Issue of October 15, 2009 © 2009 by Pontificia Universidad Catolica de Valparaiso -- Chile Received December 2, 2008 / Accepted June 29, 2009

Biocontrol capacity of wild and mutant Trichoderma harzianum (Rifai) strains on Rhizoctonia solani 618: effect of temperature and soil type during storage

Biocontrol capacity of wild and mutant Trichoderma harzianum (Rifai) strains on Rhizoctonia solani 618: effect of temperature and soil type during storage

Antifungal activity of thyme oil againstGeotrichum citri-aurantii in vitroandin vivo

To investigate antifungal effect of thyme oil on Geotrichum citri-aurantii arthroconidia germination and germ tube elongation, to reveal effects of thyme oil on morphological structures on fungal hyphae and arthroconidia and to assess potential bio-control capacities of thyme oil against disease suppression in vivo conditions. Thyme oil controlled the growth of G. citri-aurantii effectively. Arthroconidia germination and germ tube elongation in potato dextrose broth was greatly inhibited by thyme oil. At 600 microl l(-1), it inhibited the germination of about 94% of the arthroconidia and the germ tube length was only 4.32 +/- 0.28 microm. Observations using light microscope, scanning electron microscope and transmission electron microscope revealed ultrastructural modifications caused by thyme oil that included markedly shrivelled and crinkled hyphae and arthroconidia, plasma membrane disruption and mitochondrial disorganization. Thyme oil applied to 'Satsuma' mandarin oranges that had been artificially wounded and inoculated with G. citri-aurantii reduced sour rot from 78.1% among untreated control fruit to 14.1% after 5 days at 26 degrees C. Thyme oil applied to intact fruits reduced the decay from 76% among untreated control fruit to 35% after 30 days at 20 degrees C. Thyme oil treatment did not harm 'Satsuma' mandarin oranges when they were examined after treatment and storage at 20 degrees C for 30 days. Thyme oil may provide an alternative means of controlling postharvest sour rot on citrus fruit. The use of such essential oil may constitute an important alternative to synthetic fungicides. They can be exploited in commercial production and applied under storage and greenhouse conditions.

Real‐time PCR quantification and live‐cell imaging of endophytic colonization of barley (Hordeum vulgare) roots byFusarium equisetiandPochonia chlamydosporia

*New tools were developed for the study of the endophytic development of the fungal species Fusarium equiseti and Pochonia chlamydosporia in barley (Hordeum vulgare) roots. These were applied to monitor the host colonization patterns of these potential candidates for biocontrol of root pathogens. * Molecular beacons specific for either F. equiseti or P. chlamydosporia were designed and used in real-time polymerase chain reaction (PCR) quantification of fungal populations in roots. Genetic transformation of isolates with the green fluorescent protein (GFP) gene was carried out using an Agrobacterium tumefaciens-mediated transformation protocol, and spatial patterns of root colonization were investigated by laser confocal microscopy. * Quantification of endophytes by real-time PCR in roots of barley gave similar results for all fungi, and was more accurate than culturing methods. Conversely, monitoring of root colonization by GFP-expressing transformants showed differences in the endophytic behaviours of the two species, and provided evidence of a plant response against endophyte colonization. * Both F. equiseti and P. chlamydosporia colonized barley roots endophytically, escaping attempts by the host to prevent fungal growth within root tissues. This strongly supports a balanced antagonism between the virulence of the colonizing endophyte and the plant defence response. Development of real-time PCR techniques and GFP transformants of these fungal species will facilitate future work to determine their biocontrol capacity.

Biocontrol of major postharvest pathogens on apple using Rhodotorula glutinis and its effects on postharvest quality parameters

The biocontrol activity of Rhodotorula glutinis on gray mold decay and blue mold decay of apple caused by Botrytis cinerea and Penicillium expansum, respectively, was investigated, as well as its effects on postharvest quality of apple fruits. The results show there was a significant negative correlation between concentrations of the yeast cells and the disease incidence of the pathogens. The higher concentration of the R. glutinis, the better effect of the biocontrol capacity. At concentrations of R. glutinis 1 × 10 8 CFU ml −1, the amount of gray mold decay was completely inhibited after 5 days incubation at 20 °C, after challenge with B. cinerea spores suspension of 1 × 10 5 spores ml −1; While the blue mold decay was completely inhibited at concentrations of 5 × 10 8 CFU ml −1, at challenged with P. expansum spores suspension of 5 × 10 4 spores ml −1 . These results demonstrated that the efficacy of R. glutinis in controlling of gray mold decay of apples was better than the efficacy of controlling blue mold. R. glutinis within inoculated wounds on apples increased in numbers at 20 °C from an initial level of 9.5 × 10 5 CFU per wound to 2.24 × 10 7 CFU at 20 °C after 1 day. The highest population of the yeast was recovered 4 days after inoculation, the yeast population in wounds increased by 56.9 times. After that, the population of the yeast began to decline very slowly. R. glutinis significantly reduced the incidence of natural infections on intact fruit from 75% in the control fruit to 28.3% after 5 days at 20 °C, and from 58.3 to 6.7% after 30 days at 4 °C followed by 4 days at 20 °C. R. glutinis treatment had no deleterious effect on quality parameters after 5 days at 20 °C or after 30 days at 4 °C followed by 4 days at 20 °C.

Biocontrol potential and polyphasic characterization of novel native Trichoderma strains against Macrophomina phaseolina isolated from sorghum and common bean

Native strains of Trichoderma isolated from sorghum and common bean crop soils were investigated to assess their biocontrol potential over the phytopathogenic fungus Macrophomina phaseolina, isolated from diseased plants. The Trichoderma strains were characterized with a polyphasic approach, which combined the analysis of their morphological characteristics, enzymatic activity, macro- and microculture test results, rDNA restriction patterns (AFLP), ITS1-5.8S-ITS2 rDNA sequences, and protein profiles. The integration of these data sets can be used to select new isolates as biological control agents against native fungal phytopathogens. In general, we observed a positive correlation between the secretion of beta-1,3-glucanase and N-acetylhexosaminidase, and the biocontrol capacities of all the Trichoderma isolates. Strains with the best hyperparasitic behavior against M. phaseolina isolated from diseased bean and sorghum were Trichoderma sp. (TCBG-2) and Trichoderma koningiopsis (TCBG-8), respectively.

Pichia angustais an effective biocontrol yeast against postharvest decay of apple fruit caused byBotrytis cinereaandMonilia fructicola

The efficacy of eight isolates of Pichia angusta against three common postharvest pathogens of apple fruit was evaluated for the first time. All tested strains showed significant biocontrol activity against both Botrytis cinerea and Monilia fructicola, whereas efficacy against Penicillium expansum was poor. A leucine-auxotrophic mutant had no significant biocontrol activity against brown rot of apple, while the addition of 0.6-1.2 g L(-1) leucine in the fruit wound fully restored the biocontrol activity of this mutant against M. fructicola. Given the extremely well-developed classical and molecular genetics, the availability of genomic libraries, and its complete genomic sequence, this species can serve to elucidate the mechanisms related to biocontrol capacity.

Lack of biocontrol capacity in a non-pathogenic mutant of Fusarium oxysporum f. sp. melonis

The aim of this study was to assess the biocontrol capacity of rev157, a non-pathogenic mutant of a pathogenic strain of Fusarium oxysporum f. sp. melonis (Fom24). Inoculated in association with the virulent parental strain, the mutant rev157 did not protect the host plant (muskmelon) against infection by Fom24. Applied on flax, a non-host plant, the mutant rev157 was not able to protect it against its specific pathogen F. oxysporum f. sp. lini. On the contrary the parental strain Fom24 did protect flax as well as a soil-borne biocontrol strain (Fo47). Since the mutant rev157 was affected neither in its growth in vitro nor in its capacity to penetrate into the roots, it can be speculated that the mutation has affected traits responsible for interactions within the plant. In F. oxysporum the pair of strains Fom24/rev157 is a good candidate to identify genes involved in the biocontrol capacity of F. oxysporum and to test the hypothesis of a link between capacity to induce the disease and capacity to induce resistance in the plant.

EVALUATION OF TOLERANCE OF COLLETOTRICHUM ACUTATUM IN STRAWBERRY PLANTS TRANSFORMED WITH TRICHODERMA-DERIVED GENES

ISHS International Symposium on Biotechnology of Temperate Fruit Crops and Tropical Species EVALUATION OF TOLERANCE OF COLLETOTRICHUM ACUTATUM IN STRAWBERRY PLANTS TRANSFORMED WITH TRICHODERMA-DERIVED GENES