Potential Biocontrol Strains Research Articles

Identification of Meloidogyne panyuensis (Nematoda: Meloidogynidae) infecting Orah (Citrus reticulata Blanco) and its impact on rhizosphere microbial dynamics: Guangxi, China.

Root-knot nematode disease severely affects the yield and quality of the mandarin variety Citrus reticulata Blanco "Orah" in Guangxi, China. Nevertheless, the pathogen and the effects of this disease on microbial communities remain inadequately understood. This study identified the root-knot nematode Meloidogyne panyuensis in the rhizosphere of infected Orah using morphological and molecular biological methods. Soil chemical properties indicated that organic matter, total nitrogen (TN), total phosphorus (TP), available phosphorus (AP), total potassium (TK), and available potassium (AK) were significantly higher in the rhizosphere soil of M. panyuensis-infected Orah than in that of healthy plants. The relative abundance of the bacteria Bacillus, Sphingomonas, and Burkholderia-Caballeronia-Paraburkholderia, as well as the fungi Lycoperdon, Fusarium, Neocosmospora, Talaromyces, and Tetragoniomyces, was elevated in the rhizosphere soil of M. panyuensis-infected plants. Furthermore, organic matter, TN, available nitrogen (AN), TP, AP, TK, and AK exhibited positive correlationswith these bacteria and fungi in the rhizosphere soil of M. panyuensis-infected Orah. Potential biocontrol strains, such as Burkholderia spp., were identified by comparing the differences in rhizosphere microbial composition between healthy Orah and M. panyuensis-infected Orah. Our findings provide a foundation for the early warning and prevention of root-knot nematode disease in Orah.

Effect of pH on the growth and competition of Trichoderma spp. and Fusarium spp.

Collar rot caused by Fusarium spp. is a serious threat to the production of Passiflora edulis. However, biocontrol methods are lacking. Trichoderma spp., as the most widely applied biocontrol fungus, can be effective in managing crop diseases. The effectiveness is significantly influenced by environmental factors, such as soil pH. To screen potential biocontrol strains against collar rot of P. edulis, and to explore the effect of pH on the inhibition rate of Trichoderma spp., we selected four Trichoderma species and four Fusarium species isolated from P. edulis planting area in Xishuangbanna. The growth dynamics of different strains under different pH conditions were determined using the mycelial growth rate method. The effect of pH on the growth inhibition of Fusarium spp. by Trichoderma spp. was investigated using the plate confrontation assay. The results showed that the optimal growth pH range was 4-6 for Trichoderma spp. and 7-9 for Fusarium spp. All four Trichoderma strains exhibited significant inhibitory effects on the growth of the four Fusarium strains. T. harzianum showed the most notable inhibition, reaching up to a 72% inhibitory rate. Moreover, pH significantly influenced the inhibitory effect of Trichoderma spp., with variations observed depending on the specific species of Trichoderma spp. and Fusarium spp. Therefore, it is essential to consider the environmental pH impact on the efficacy of biocontrol agents when applying biological control measures in the field, tailored to the specific pathogen and biocontrol agent involved.

Seasonal variations in the composition and diversity of rhizosphere soil microbiome of bamboo plants as infected by soil-borne pathogen and screening of associated antagonistic strains

As the occurrence and severity of soil-borne diseases are strongly season-dependent, the ecological processes that shift plant microbiome compositions and functions upon pathogen infection in different seasons are rarely studied. In this study, we compared the differences in fungal and bacterial communities in rhizosphere soil between healthy and diseased bamboo plants throughout the four seasons at the main bamboo production site in China and investigated how basal rot disease caused by Fusarium proliferatum affected the composition, structure and ecological functions of rhizosphere-associated microorganisms. The results showed that diseased bamboo plants enriched the highest abundance of Fusarium genus in rhizosphere soil while receiving largely increased stem lesion area in summer. Microbiome analysis further proved that healthy bamboo plants harbored more ecologically beneficial microbial taxa that might function as biological protectants in their rhizosphere soil than diseased plants at the same season. Three individual strains with biocontrol potentials were further isolated and identified as Trichoderma spirale, Bacillus velezensis and Streptomyces mirabilis. The antagonistic tests further demonstrated their inhibition effects on the growth and pathogenicity of the pathogenic fungus F. proliferatum in vitro and in vivo. This study advances our understanding of soil-borne pathogen mediated changes in microbiome structure and function in plant rhizosphere soil and provides potential biocontrol strains for increasing plant resistance against basal rot disease.

Control of Salmonella in mung bean sprouts by antagonistic spore-forming Bacilli

Sprouting seeds are considered part of a healthy and nutritious diet, and accordingly, their consumption increases globally. However, edible sprouts are occasionally linked to outbreaks of foodborne diseases, with Salmonella enterica being a frequent contaminant. Biocontrol was recently proposed as a possible approach to combat sprout contamination during production. The purpose of this study was to identify sprout-derived Salmonella-antagonists as a basis for the development of new biocontrol agents. To this end, spore-forming bacteria were isolated from sprouted mung bean seeds, based on either in vitro anti-Salmonella activity or rough colony morphology and tested in planta for inhibition of mung bean sprout colonization. Artificially contaminated seeds (2 log CFU/g) treated with selected Bacillus strains (7 log CFU/ml) demonstrated extensive inhibition of Salmonella growth after 4-days of sprouting, resulting in 3.3–5.4 log CFU/g reduction compared to control (untreated). The potent antagonistic activity of the selected Bacillus isolates was confirmed for different S. enterica serovars. Interestingly, some of the antagonistic strains that demonstrated activity in vitro were not active in planta and vice versa, indicating that in vitro testing may not be the best strategy for screening potential biocontrol strains in sprouts. Confocal microscopy imaging demonstrated that in the absence of Bacillus, Salmonella readily proliferates and colonizes the entire sprouting seed. Treatment with an antagonistic Bacillus isolate markedly inhibited the colonization of Salmonella. Scanning electron microscopy of Bacillus-treated sprouts demonstrated the colonization of the sprout with biofilm-like structures, indicating the activation of the biofilm formation mechanism during sprout colonization. The identified Bacillus isolates provide a basis for the future development of novel biocontrol agents against Salmonella and perhaps other foodborne pathogens in edible sprouts.

Insight into Biological Control Potential of Hirsutella citriformis against Asian Citrus Psyllid as a Vector of Citrus Huanglongbing Disease in America.

Studies on Hirsutella citriformis Speare are scarce. Among these, some reports have focused on phenotypic identification, based on its morphological structure and morphometric characteristics. This fungus is known to control economically important citrus crop pests. In recent years, H. citriformis has received increased attention as a control agent for the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae), which causes the Huanglongbing (HLB) disease. Unfortunately, formal H. citriformis strains characterization is marginal, which mainly involves the role of biologically active exudates (metabolites) produced during their growth. Information regarding their mode of action and biocontrol potential is limited. However, epizootics reports of this fungus, under suitable environmental conditions for its development (25 °C to 28 °C and ~80% relative humidity), have demonstrated its parasitization efficacy. Therefore, it becomes challenging to determine whether H. citriformis strains may be developed as commercial products. In this review, we showed relevant information on isolation and bioassay strategies of H. citriformis to evaluate potential biocontrol strains under laboratory and field conditions in America.

Biocontrol properties from phyllospheric bacteria isolated from Solanum lycopersicum and Lactuca sativa and genome mining of antimicrobial gene clusters

BackgroundBiocontrol agents are sustainable eco-friendly alternatives for chemical pesticides that cause adverse effects in the environment and toxicity in animals including humans. An improved understanding of the phyllosphere microbiology is of vital importance for biocontrol development. Most studies have been directed towards beneficial plant-microbe interactions and ignore the pathogens that might affect humans when consuming vegetables. In this study we extended this perspective and investigated potential biocontrol strains isolated from tomato and lettuce phyllosphere that can promote plant growth and potentially antagonize human pathogens as well as plant pathogens. Subsequently, we mined into their genomes for discovery of antimicrobial biosynthetic gene clusters (BGCs), that will be further characterized.ResultsThe antimicrobial activity of 69 newly isolated strains from a healthy tomato and lettuce phyllosphere against several plant and human pathogens was screened. Three strains with the highest antimicrobial activity were selected and characterized (Bacillus subtilis STRP31, Bacillus velezensis SPL51, and Paenibacillus sp. PL91). All three strains showed a plant growth promotion effect on tomato and lettuce. In addition, genome mining of the selected isolates showed the presence of a large variety of biosynthetic gene clusters. A total of 35 BGCs were identified, of which several are already known, but also some putative novel ones were identified. Further analysis revealed that among the novel BGCs, one previously unidentified NRPS and two bacteriocins are encoded, the gene clusters of which were analyzed in more depth.ConclusionsThree recently isolated strains of the Bacillus genus were identified that have high antagonistic activity against lettuce and tomato plant pathogens. Known and unknown antimicrobial BGCs were identified in these antagonistic bacterial isolates, indicating their potential to be used as biocontrol agents. Our study serves as a strong incentive for subsequent purification and characterization of novel antimicrobial compounds that are important for biocontrol.

Isolation, identification, and assessment of soil bacteria as biocontrol agents of pea root rot caused by Aphanomyces euteiches

Aphanomyces euteiches is a soil-borne pathogen that causes root rot of pea and can significantly affect pea production in western Canada. This study aimed to isolate and identify soil bacteria with antagonistic activity towards A. euteiches mycelial and zoospore developmental stages under in vitro conditions and assess their potential as biocontrol agents against aphanomyces root rot in field pea under growth chamber conditions. In vitro screening of soil bacteria identified 184 antagonistic isolates, including 22 from an existing culture collection. Mean mycelial growth inhibition zones ranged from 1 to 12 mm, and mean zoospore germination inhibition ranged from 0% to 100%. Use of 16S rDNA sequence analysis placed isolates into 18 different bacterial genera. Screening of 47 bacteria that inhibited both infective stages identified 29 potential biocontrol strains, including Rhizobium spp. that significantly (α = 0.05) suppressed aphanomyces root rot in field pea grown in vermiculite, suggesting the intriguing possibility of using N-fixing Rhizobium inoculants as biocontrol agents for aphanomyces control. Further screening of 20 isolates as soil inoculants identified K-Hf-L9 (Pseudomonas fluorescens), PSV1-7 (Pantoea agglomerans), and K-Hf-H2 (Lysobacter capsici) isolates as having the highest biocontrol activity, significantly (α = 0.05) suppressing aphanomyces root rot in field pea in growth chamber trials. This study demonstrates the possibility of aphanomyces root rot management using biocontrol agents.

Biocontrol impact of AHL degrading actinobacteria on quorum sensing regulated virulence of phytopathogen Pectobacterium carotovorum subsp. carotovorum BR1

The aim of this study was to isolate soilborne AHL degrading actinobacteria that can be used as biocontrol agents against the quorum sensing mediated virulence of Pectobacterium carotovorum subsp. carotovorum BR1 in in vitro and in planta studies. Actinobacterial strains capable of N-acyl homoserine lactone (AHL) degradation, Glutamicibacter nicotianae AI5a and Rhodococcus pyridinivorans AI4 were isolated and screened using Chromobacterium violaceum CV026 as a biosensor in an AHL degradation bioassay. The effect of AHL degradation by the potential biocontrol strains was studied by a co-culture assay. Reduction in plant cell wall degrading enzymes produced by PccBR1 due to the quorum quenching activity of the potential biocontrol strains was studied using colorimetric assays. The ability of the isolates to reduce soft rot symptoms was studied in in vitro cucumber and potato maceration assays. A cucumber plant infection model was developed to study blackleg in greenhouse experiments. G. nicotianae AI5a, R. pyridinivorans AI4 and R. erythropolis CRD13.3C were able to reduce 3-oxo-C6- N-acyl homoserine lactone and production of plant cell wall degrading enzymes by PccBR1. The isolates were also able to prevent and attenuate soft rot caused by PccBR1 in vitro in potato and cucumber maceration assays. The isolates were also able to prevent blackleg in an in planta cucumber infection model. Among the three actinobacterial isolates, G. nicotianae AI5a showed best results and has immense potential as a biocontrol agent against the virulence of the quorum sensing phytopathogen PccBR1.

Isolation of Bacillus subtilis strain SEM-2 from silkworm excrement and characterisation of its antagonistic effect against Fusarium spp.

Fusarium wilt is a devastating soil-borne disease mainly caused by highly host-specific formae speciales of Fusarium spp. Antagonistic microorganisms play a very important role in Fusarium wilt control. Isolation of potential biocontrol strains has become increasingly important. Bacterial strain SEM-2 was isolated from the high-temperature stage of silkworm excrement composting. SEM-2 exhibited a considerable antagonistic effect against Fusarium graminearum mycelial growth and spore germination. The results of pot experiments suggested that SEM-2 has a better inhibitory effect on the early stage of disease occurrence. The green fluorescent protein labelled SEM-2 coated on the surface of tomato seeds colonised the roots of tomato plants in 15 days. Genome sequencing identified SEM-2 as a new strain of Bacillus subtilis, and genome annotation and analysis determined gene clusters related to the biosynthesis of antimicrobials, such as bacillaene, fengycin, bacillibactin, subtilosin A, surfactin, and bacilysin. Interestingly, liquid chromatography - quadrupole time-of-flight mass spectrometry revealed that metabolites in pathways associated with the synthesis of secondary metabolites and antibiotics were highly differentially expressed. These findings may help to explain the mode of action of B. subtilis SEM-2 against Fusarium spp.

Characterization and sequence analysis of potential biofertilizer and biocontrol agent Bacillus subtilis strain SEM-9 from silkworm excrement.

Fusarium wilt is a devastating soil-borne disease caused mainly by highly host-specific formae speciales of Fusarium oxysporum. Antagonistic microorganisms play a very important role in Fusarium wilt control, and the isolation of potential biocontrol strains is becoming more and more important. We isolated a bacterial strain (SEM-9) from the high-temperature stage of silkworm excrement composting, which had a marked ability to solubilize phosphorus, promote the growth and increase the yield of the small Chinese cabbage, and which also exhibited considerable antagonistic effect towards Fusarium sambucinum and other fungi. The result of physiological and biochemical analyses, as well as genome sequencing, showed that SEM-9 was a strain of Bacillus subtilis. Through genome annotation and analysis, it was found that SEM-9 contained genes related to the regulation of biofilm formation, which may play an important role in colonization, and gene clusters encoding the biosynthesis of antimicrobials, such as surfactin, bacilysin, fengycin, and subtilosin-A. The production of such antifungal compounds may constitute the basis of the mode-of-action of SEM-9 against Fusarium spp. These data suggested that the SEM-9 strain has potential as both a biofertilizer and a biocontrol agent, with the potential to manage Fusarium wilt disease in crops.

Microbe‐mediated control of Aspergillus flavus in stored rice grains with a focus on aflatoxin inhibition and biodegradation

AbstractBiological control of mycotoxigenic fungi using antagonistic microbes is a promising alternative to agricultural chemicals for postharvest storage. In this study, we evaluated rice‐derived bacterial strains to identify biocontrol agents to inhibit Aspergillus flavus in stored rice grains. Consequently, we obtained three potential biocontrol strains (Microbacterium testaceum KU313, Bacillus megaterium KU143 and Pseudomonas protegens AS15) from 26 tested strains that were prescreened from the 460 strains isolated from rice grains. The three selected strains proved to be effective biocontrol agents showing antifungal activity against A. flavus and good colonisation ability on rice grains, along with inhibition of the fungal growth and aflatoxin production. In particular, P. protegens AS15 greatly inhibited the aflatoxins produced by A. flavus on rice grains to 8.68 (percent aflatoxin reduction relative to control = 82.9%) and 18.05 (68.3 %) ng g−1 dry weight of rice grains, compared with the 50.89 and 56.97 ng g−1 dry weight of rice grains of the MgSO4 control at 1 and 2 weeks after inoculation, respectively. In addition, strain AS15 had a significant ability to not only degrade aflatoxin B1 (the most harmful aflatoxin), but also utilise the toxin for bacterial growth in a nutrient‐deficient medium. Therefore, the selected bacterial strains could be environmentally sound alternatives for the management of A. flavus and aflatoxin production by reducing the fungal damage to stored rice grains. This would also reduce the human and animal health hazards associated with the consumption of fungus‐contaminated rice grains. To our knowledge, this is the first report of the potential of the bacterial species M. testaceum and P. protegens as biocontrol agents for controlling aflatoxigenic A. flavus on stored rice grains.

The Profile and Antimicrobial Activity of Bacillus Lipopeptide Extracts of Five Potential Biocontrol Strains.

In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains-ethyl acetate extraction, and acid precipitation followed by methanol extraction—was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins, and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1, and SS-38.4 showed the highest inhibition zones. An iturin analog is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2, and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.

Potential biocontrol Bacillus sp. strains isolated by an improved method from vinegar waste compost exhibit antibiosis against fungal pathogens and promote growth of cucumbers

An in vitro antagonism test is a typical procedure for the selection of potential biocontrol strains. However, the traditional method of screening antagonistic bacteria in vitro is a time consuming method when conducting large-scale screening trials. In this study, an improved method for the selection of antagonistic bacteria in vitro from compost was established based on the traditional method. 21 Antagonistic bacteria out of 33 target strains isolated from vinegar waste compost using the improved method. The 16S rDNA gene showed the 21 strains all belonged to the Bacillus genus and 18 different types of fingerprints were obtained by enterobacterial repetitive inter-genic consensus (ERIC)-PCR. 18 Selected strains which had the unique fingerprints all exhibited broad-spectrum antagonism towards the tested fungi and at least two enzyme activities in vitro. Among them, majority of the isolates were siderophore producer, some of them showed nitrogen-fixing ability and small of them were IAA producer. Four out of five selected strains were found both to be effective in controlling wilt and damping-off disease and four strains showed strong growth-promoting activities for cucumber seedlings under greenhouse conditions. Thus, these results demonstrated that the improved method was an effective and rapid means to screen potential antagonistic microorganisms in vitro. The results also showed that Bacillus sp. strains in vinegar waste compost exhibited antibiosis against fungal pathogens and promoted the growth of cucumber seedlings.

Biocontrol of Phytophthora Blight and Anthracnose in Pepper by Sequentially Selected Antagonistic Rhizobacteria against Phytophthora capsici.

We previously developed a sequential screening procedure to select antagonistic bacterial strains against Phytophthora capsici in pepper plants. In this study, we used a modified screening procedure to select effective biocontrol strains against P. capsici; we evaluated the effect of selected strains on Phytophthora blight and anthracnose occurrence and fruit yield in pepper plants under field and plastic house conditions from 2007 to 2009. We selected four potential biocontrol strains (Pseudomonas otitidis YJR27, P. putida YJR92, Tsukamurella tyrosinosolvens YJR102, and Novosphingobium capsulatum YJR107) among 239 bacterial strains. In the 3-year field tests, all the selected strains significantly (P < 0.05) reduced Phytophthora blight without influencing rhizosphere microbial populations; they showed similar or better levels of disease suppressions than in metalaxyl treatment in the 2007 and 2009 tests, but not in the 2008 test. In the 2-year plastic house tests, all the selected strains significantly (P < 0.05) reduced anthracnose incidence in at least one of the test years, but their biocontrol activities were variable. In addition, strains YJR27, YJR92, and YJR102, in certain harvests, increased pepper fruit numbers in field tests and red fruit weights in plastic house tests. Taken together, these results indicate that the screening procedure is rapid and reliable for the selection of potential biocontrol strains against P. capsici in pepper plants. In addition, these selected strains exhibited biocontrol activities against anthracnose, and some of the strains showed plant growth-promotion activities on pepper fruit.

The Caenorhabditis elegans assay: a tool to evaluate the pathogenic potential of bacterial biocontrol agents

Bacterial biocontrol agents (BCAs) open up the possibility of controlling plant pathogens in an environmentally friendly way. Although they are naturally occurring microbes, some of them can cause diseases in humans. For successful registration it is necessary to test potentially adverse effects on the human health of at-risk candidates. Existing pathogenicity assays are cost-intensive, time-consuming and furthermore they are often inappropriate for facultative pathogens. We developed a new, fast and inexpensive bioassay on the basis of the nematode Caenorhabditis elegans, which is a well-accepted model organism to study bacterial pathogenicity. A selection of eight strains from clinical and environmental origin as well as potential and commercial BCAs from the genera Bacillus, Pseudomonas, Serratia and Stenotrophomonas were screened for their potential to kill the nematode in an in vitro agar plate assay. Furthermore, the motility and reproductive behaviour of nematodes exposed to strains were tested in comparison with those fed by the human pathogen Pseudomonas aeruginosa QC14-3-8 (positive control) and the negative control Escherichia coli OP50. Commercial as well as potential biocontrol strains did not display any adverse effects in all tests. In contrast, the C. elegans assay showed slight effects for clinical and environmental Stenotrophomonas strains. Results showed that the nematode C. elegans provides a model system to indicate the pathogenic potential of BCAs in a very early stage of product development.

Genetic diversity shown in Trichoderma biocontrol isolates

Genetic variability within 69 biocontrol isolates of Trichoderma, obtained from different geographical locations and culture collections and selected as biocontrol agents, was studied. Sequence data, obtained from the ITS1 region of rDNA and a fragment of the translation elongation factor 1 (tef1) gene, were used in a phylogenetic analysis. Phylograms showing similar topologies were generated using alignments containing the ITS1 region or a portion of the tef1 gene. 21 distinct ITS1 sequence types and 17 distinct tef1 sequence types were identified among the 69 isolates. More than 50% of the potential biocontrol strains were grouped within Trichoderma sect. Pachybasium; of these, 81% were grouped within the cluster that included the ex-type strains of T. harzianum and T. inhamatum, and 16% were grouped with T. virens. Within T. sect. Trichoderma, which included 36% of the 69 strains, 56% were grouped with T. asperellum, and 24% with T. viride, T. atroviride or T. koningii. Only 10% of the strains studied were located in T. sect. Longibrachiatum.

Biological and molecular characterisation of potential biocontrol strains of Trichoderma.

Thirty-four strains of seven species of Trichoderma isolated from various fungal sources were compared for direct mycoparasitic activity (MPA), chitinase production and antibiotic activity (ANA) in order to choose the most appropriate partners for a strain-breeding programme. Within species genetic differences were also assesses in T. hamatum, T. harzianum and T. viride by means of random amplification of polymorphic DNA (RAPD). Endochitinase activities of the Trichoderma strains ranged between 20.4 and 1264.5 units/g dry weight of mycelium. The correlation between MPA and chitinase activity was not unambiguous and no correlation existed between MPA and ANA. The RAPD patterns of T. viride strains were highly variable, while isolates of T. harzianum proved to be more uniform; T. hamatum revealed remarkable intraspecific divergence. All these three comprised certain pairs of strains that are promising participants of a strain-improving programme, since their strong genetic affinities offer good changes for combining their contrasted biocontrol traits.

Differentiation of six strains of Bacillus thuringiensis by hydrolyzable fatty acid composition

Six potential biocontrol strains of Bacillus thuringienis grown under identical conditions on fattyacid-free King's B medium were differentiated on the basis of their hydrolyzable fatty acid content. Thirty-four compounds were identified as their methyl esters in hydrolysates, among which the major fatty acids were i-15.0 (13.7-23.2%), i-13:0 (6.8-10.7%), i-14:0 (5.0-7.7%, 14:0 (3.0-4.1%), a-15:0 (3.9-9.9%), i-16:0 (3.6-7.2%), 16:0 (3.2-11.6%), i-17:0 (3.2-9.8%), 18:0 (tr-13.5%), a monounsaturated C 16 acid (4.5-9.9%), a monounsaturated branched C 17 acid (2.8-5.8), a diunsaturated C 18 acid (0-5.8%), and a monounsaturated C 18 acid (0.3-4.8%)