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A new isolate of the nematophagous fungus Duddingtonia flagrans a biological control agent against free-living larvae of horse strongyles.

An experiment was carried out in 1997 to test the efficacy of an isolate of the microfungus Duddingtonia flagrans against free-living stages of horse strongyles under conditions in the field and to assess the eventual effect of the fungus on the normal degradation of faeces. Faecal pats were made from faeces of a naturally strongyle infected horse, which had been fed fungal material at a dose level of 106 fungal unit/kg bwt. Control pats without fungi were made from faeces collected from the same animal just before being fed fungi. Faecal cultures set up for both groups of faeces to monitor the activity of the fungus under laboratory conditions showed that the fungus significantly reduced the number of infective third-stage larvae (L3) by an average of 98.4%. Five faecal pats from each batch of faeces were deposited on pasture plots at 3 times during spring-summer. The herbage around each pat was sampled fortnightly to recover L3 transmitted from faeces. The results showed that the herbage infectivity around fungus-treated pats was reduced by 85.8-99.4%. The remaining faecal material at the end of each sampling period was collected, and the surviving L3 were extracted. Significantly fewer larvae were recovered from the fungus-treated pats. Analysis of wet and dry weight of the collected pats, as well as their organic matter content, were performed to compare the degradation of faeces of both groups. The results indicated that the presence of the fungus did not alter the degradation of the faeces.

Animal Feed↗

Expression of the mosquitocidal toxins of Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis by recombinant Caulobacter crescentus, a vehicle for biological control of aquatic insect larvae.

In the quest for effective control of mosquitoes, attention has turned increasingly to strains of the bacteria Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis, which produce potent toxins with specific mosquitocidal activities. However, sedimentation of the bacterial spores limits the duration of effective control after field application of these bacilli. We describe here the cloning of genes encoding the 51.4- and 41.9-kDa toxins from B. sphaericus 2297, the 100-kDa toxin from B. sphaericus SSII-1, and the 130-kDa toxin from B. thuringiensis subsp. israelensis into the broad-host-range plasmid pRK248 and the transfer of these genes for expression in Caulobacter crescentus CB15. The recombinant C. crescentus cells were shown to be toxic to mosquito larvae. Caulobacter species are ubiquitous microorganisms residing in the upper regions of aquatic environments and therefore provide the potential for prolonged control by maintaining mosquitocidal toxins in larval feeding zones.

Animals↗

Iron-binding compounds from Agrobacterium spp.: biological control strain Agrobacterium rhizogenes K84 produces a hydroxamate siderophore.

Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A. tumefaciens, A. rhizogenes, and A. vitis. The crown gall biocontrol agent A. rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts. Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium. Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media. Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84. One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions. Thus, the hydroxamate compound has siderophore activity. A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84. The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84. A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium. This cosmid clone contained the region in which Tn5 was located in the mutant. Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily. Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria. Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A. rhizogenes that produce hydroxamate-type compounds under low-iron conditions. Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.

Amino Acid Sequence↗

Fate of the biological control agent Pseudomonas aureofaciens TX-1 after application to turfgrass.

The fate and impact of Pseudomonas aureofaciens TX-1 following application as a biocontrol agent for fungi in turfgrass were studied. The organism was applied with a modified irrigation system by using a preparation containing 1 x 10(6) P. aureofaciens TX-1 CFU ml(-1) about 100 times between May and August. We examined the impact of this repeated introduction of P. aureofaciens TX-1 (which is known to produce the antimicrobial compound phenazine-1-carboxylic acid) on the indigenous microbial community of the turfgrass system and on establishment of introduced bacteria in the soil system. A PCR primer-DNA hybridization probe combination was developed to accurately monitor the fate of P. aureofaciens TX-1 following application in irrigation water. To assess the impact of frequent P. aureofaciens TX-1 applications on the indigenous bacterial community, turfgrass canopy, thatch, and rhizosphere samples were obtained during the growing season from control and treated plots and subjected to DNA extraction procedures and denaturing gradient gel electrophoresis (DGGE). PCR amplification and hybridization of extracted DNA with the P. aureofaciens TX-1-specific primer-probe combination revealed that P. aureofaciens TX-1 not only became established in the rhizosphere and thatch but also was capable of overwintering. Separation of PCR-amplified partial 16S rRNA genes by DGGE showed that the repeated application of P. aureofaciens TX-1 in irrigation water resulted in transient displacement of a leaf surface bacterial community member. There was no obvious alteration of any dominant members of the thatch and rhizosphere microbial communities.

DNA, Ribosomal↗

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control.

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.

Amino Acid Sequence↗

Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens.

Pseudomonas fluorescens 2-79RN(10) protects wheat against take-all disease caused by Gaeumannomyces graminis var. tritici; however, the level of protection in the field varies from site to site. Identification of soil factors that exert the greatest influence on disease suppression is essential to improving biocontrol. In order to assess the relative importance of 28 soil properties on take-all suppression, seeds were treated with strain 2-79RN(10) (which produces phenazine-1-carboxylate [PCA(+)]) or a series of mutants with PCA(+) and PCA(-) phenotypes. Bacterized seeds were planted in 10 soils, representative of the wheat-growing region in the Pacific Northwest. Sixteen soil properties were correlated with disease suppression. Biocontrol activity of PCA(+) strains was positively correlated with ammonium-nitrogen, percent sand, soil pH, sodium (extractable and soluble), sulfate-sulfur, and zinc. In contrast, biocontrol was negatively correlated with cation-exchange capacity (CEC), exchangeable acidity, iron, manganese, percent clay, percent organic matter (OM), percent silt, total carbon, and total nitrogen. Principal component factor analysis of the 16 soil properties identified a three-component solution that accounted for 87 percent of the variance in disease rating (biocontrol). A model was identified with step-wise regression analysis (R(2) = 0.96; Cp statistic = 6.17) that included six key soil properties: ammonium-nitrogen, CEC, iron, percent silt, soil pH, and zinc. As predicted by our regression model, the biocontrol activity of 2-79RN(10) was improved by amending a soil low in Zn with 50 micro g of zinc-EDTA/g of soil. We then investigated the negative correlation of OM with disease suppression and found that addition of OM (as wheat straw) at rates typical of high-OM soils significantly reduced biocontrol activity of 2-79RN(10).

Ascomycota↗

Molecular characterization of Brevibacillus laterosporus and its potential use in biological control.

Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species.

Animals↗

Positive autoregulation and signaling properties of pyoluteorin, an antibiotic produced by the biological control organism Pseudomonas fluorescens Pf-5.

Pseudomonas fluorescens Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens. Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol. We provide evidence that pyoluteorin production is influenced by positive autoregulation. Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production. In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5. For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene (inaZ) to three separate pyoluteorin biosynthetic genes. The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments. In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5. These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.

Anti-Bacterial Agents↗

Probiotic bacteria as biological control agents in aquaculture.

There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.

Animals↗

Mosquitocidal toxins of bacilli and their genetic manipulation for effective biological control of mosquitoes.

The identification, cloning, and characterization of protein toxins from various species of bacilli have demonstrated the existence of mosquitocidal toxins with different structures, mechanisms of action, and host ranges. A start has been made in understanding the polypeptide determinants of toxicity and insecticidal activity, and the purification of toxins from recombinant organisms may lead to the elucidation of their X-ray crystal structures and the cloning of brush border membrane receptors. The results of cloning mosquitocidal toxins in heterologous microorganisms show the potential of expanding the range of susceptible mosquito species by combining several toxins of different host specificity in one cell. Toxins have been expressed in new microorganisms with the potential for increasing potency by persisting at the larval feeding zone. The powerful tools of bacterial genetics are being applied to engineer genetically stable, persistent toxin expression and expand the insecticidal host ranges of Bacillus sphaericus and Bacillus thuringiensis strains. These techniques, together with modern formulation technology, should eventually lead to the construction of mosquitocidal microorganisms which are effective enough to have a real impact on mosquito-borne diseases.

Animals↗

Competition and antibiosis in the biological control of potato scab.

Nonpathogenic, antibiotic-producing streptomycetes have been shown to reduce potato scab when added to disease-conducive soil. Spontaneous mutants of the pathogenic Streptomyces scabies RB4 that are resistant to at least one antibiotic activity produced by the nonpathogenic suppressive isolates Streptomyces diastatochromogenes strain PonSSII and S. scabies PonR have been isolated. To determine the importance of antibiosis in this biocontrol system, these mutants were investigated for their ability to cause disease in the presence of the two pathogen antagonists in a greenhouse assay. Disease caused by one of the mutant strains was reduced in the presence of both suppressive isolates, whereas disease caused by the other five mutants was not significantly reduced by either suppressive strain. In addition, a nonpathogenic mutant of S. scabies RB4 was isolated, which produced no detectable in vitro antibiotic activity and reduced disease caused by its pathogenic parent strain when the pathogen and mutant were coinoculated into soil. Population densities of the pathogen were consistently lower than those of the suppressive strains when individual strains were inoculated into soil. When a pathogen was coinoculated with a suppressive strain, the total streptomycete population density in the pot was always less than that observed when the suppressive isolate was inoculated alone. When the pathogens were inoculated individually into soil, a positive correlation was seen between population density and disease severity. In coinoculation experiments with pathogen and suppressive strains, higher total streptomycete population densities were correlated with lower amounts of disease.

Anti-Bacterial Agents↗

Effect of genotype and root colonization in biological control of fusarium wilts in pigeonpea and chickpea by Pseudomonas aeruginosa PNA1.

Pseudomonas aeruginosa PNA1, an isolate from chickpea rhizosphere in India, protected pigeonpea and chickpea plants from fusarium wilt disease, which is caused by Fusarium oxysporum f.sp. ciceris and Fusarium udum. Inoculation with strain PNA1 significantly reduced the incidence of fusarium wilt in pigeonpea and chickpea on both susceptible and moderately tolerant genotypes. However, strain PNA1 protected the plants from fusarium wilt until maturity only in moderately tolerant genotypes of pigeonpea and chickpea. Root colonization of pigeonpea and chickpea, which was measured using a lacZ-marked strain of PNA1, showed tenfold lower root colonization of susceptible genotypes than that of moderately tolerant genotypes, indicating that this plant-bacteria interaction could be important for disease suppression in this plant. Strain PNA1 produced two phenazine antibiotics, phenazine-1-carboxylic acid and oxychlororaphin, in vitro. Its Tn5 mutants (FM29 and FM13), which were deficient in phenazine production, caused a reduction or loss of wilt disease suppression in vivo. Hence, phenazine production by PNA1 also contributed to the biocontrol of fusarium wilt diseases in pigeonpea and chickpea.

Cajanus↗

Whole cells of Bacillus subtilis AF 1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust.

In foliar and postharvest biocontrol systems, the use of active metabolites produced by antagonistic microorganisms is advantageous compared with the use of living microorganisms. Chitinases, a major group of hydrolytic enzymes produced by biocontrol agents, are involved in the lysis of cell walls of pathogenic fungi. In the present study, an attempt was made to test the partially purified beta-1,4-N-acetylglucosaminidase (NAGase) of a biocontrol strain Bacillus subtilis AF 1 for control of rust in groundnut (caused by Puccinia arachidis) and soft rot in lemons (caused by Aspergillus niger). Four proteins of molecular mass 67, 40, 37, and 32 kDa were isolated from the culture filtrates of AF 1 by affinity chromatography, of which the 67-kDa protein has detectable chitinolytic ability. This protein (NAGase) effectively inhibited the in vitro growth of A. niger in microtitre plates. In the presence of NAGase, germination of urediniospores of P. arachidis was reduced by 96% compared with the control. In a detached leaf bioassay, NAGase reduced the rust lesion frequency by >60%. NAGase significantly reduced the incidence of soft rot in harvested lemon fruits. However, fresh cells and (or) alginate formulation of AF 1 were more effective than NAGase in control of both of the test plant - pathogen systems.

Acetylglucosaminidase↗

Biological control of collar rot disease with broad-spectrum antifungal bacteria associated with groundnut.

Bacteria associated with 6 habitats of groundnut were evaluated for their broad-spectrum antifungal activity and suppression of collar rot (Aspergillus niger) of groundnut. Three hundred and ninety-three strains were tested against 8 fungal pathogens of groundnut including 5 necrotrophic fungi, Aspergillus flavus, A. niger, Rhizoctonia bataticola, Rhizoctonia solani, and Sclerotium rolfsii, and 3 biotrophic fungi, Cercospora arachidicola, Phaeoisariopsis personata, and Puccinia arachidis. Pseudomonas sp. GRS 175, Pseudomonas aeruginosa GPS 21, GSE 18, GSE 19, and GSE 30, and their cell-free culture filtrates were highly antagonistic to all the test fungi. The cell-free culture filtrates of these bacteria were fungicidal and induced mycelial deformations including hyphal bulging and vacuolization in necrotrophic fungi. The cell-free culture filtrates at 10% (v/v) concentration significantly inhibited the spore germination of biotrophic fungi. In the greenhouse, P. aeruginosa GSE 18 emerged as an effective biocontrol agent of collar rot closely followed by P. aeruginosa GSE 19. The bacterium applied as a seed treatment reduced the pre-emergence rotting and postemergence wilting by > 60%. Pseudomonas aeruginosa GSE 18 effectively colonized the groundnut rhizosphere, both in native and in A. niger infested potting mixtures. Ninety-day-old peat formulation of P. aeruginosa GSE 18 had biocontrol ability comparable with the midlog-phase cells. Pseudomonas aeruginosa GSE 18, tolerant to thiram, in combination with the fungicide had an improved collar rot control. The present study was a successful attempt in selection of broad-spectrum and fungicide tolerant biocontrol agents that can be a useful component of integrated management of collar rot.

Antibiosis↗