Control Of Plant Pathogenic Fungi Research Articles

PAENIBACILLUS POLYMYXA: ANTIBIOTICS, HYDROLYTIC ENZYMES AND HAZARD ASSESSMENT

SUMMARY Certain Paenibacillus polymyxa strains that associate with many plant species have been used effectively in the control of plant pathogenic fungi and bacteria. In this article we review the possible mechanism of action by which P. polymyxa promotes plant growth and suppresses some plant diseases. Furthermore we present an updated summary of antibiotics, autolysis, hydrolytic and autolytic enzymes and levanase produced by this bacterium. Some hazards and mild pathogenic effects are also reported, but these appear to be strain-specific and negligible. The association between plants and P. polymyxa seems to be specific and to involve co-adaptation processes. There is every reason to believe that gaining an improved understanding of these processes will enhance and facilitate efforts to wean off farmers dependence on a wide range of agricultural chemicals.

Molecular basis of resistance to cytochromebc1inhibitors

Inhibitors of the mitochondrial respiratory chain enzyme cytochrome bc1 (respiratory complex III) have been developed as antimicrobial agents. They are used in agriculture to control plant pathogenic fungi and in medicine against human pathogens, such as the malaria parasite Plasmodium falciparum, or Pneumocystis jiroveci (an opportunistic pathogenic fungus life-threatening in immuno-compromised patients). These respiratory inhibitors are thus effective against a broad range of important pathogens. Unfortunately, the problem of acquired resistance has rapidly emerged. A growing number of pathogen isolates resistant to inhibitor treatment have been reported, and this resistance is often linked to mutation within cytochrome b, one of the essential catalytic subunits of the complex. Saccharomyces cerevisiae is an invaluable model in order to assess the impact of the mutations on the sensitivity to the drugs, on the respiratory capacity and the fitness of cells. In this minireview, the inhibitors, their mode of action, and the mutations implicated in resistance and studied in yeast are briefly reviewed. Four mutations that are of particular importance in medicine and in agriculture are briefly reviewed and described in more detail and the molecular basis of resistance and of evolution of the mutations is discussed succinctly.

Identification of two group A chitinase genes in Botrytis cinerea which are differentially induced by exogenous chitin

Chitin-degrading enzymes represent potential targets for pesticides in the control of plant pathogenic fungi. Here we describe the cloning, molecular characterization, and expression analysis of two putative chitinases of Botrytis cinerea, a pathogenic fungus infecting a wide range of plants. On the basis of conserved motifs from family 18 of the glycosyl hydrolases and group A of the fungal chitinases, two fragments ( BcchiA and BcchiB) were cloned and sequenced. Expression of BcchiA and BcchiB chitinase genes upon growth under different conditions was analysed using RT-PCR. We observed that BcchiA expression was suppressed by glucose, whereas it was strongly stimulated in the presence of chitin or chitin degradation products. Conversely, BcchiB expression was not suppressed by glucose and was not stimulated by chitin or chitin degradation products. The difference in expression regulation is indicative of a functional divergence between the two chitinase paralogous genes.

Using Serratia plymuthica to control fungal pathogens of plants.

Abstract Interest in biological control of plant pathogens has increased in recent years fuelled by trends in agriculture towards greater sustainability and public concerns over the use of hazardous pesticides in the environment. Most studies on the biological control of fungal plant pathogens have tended to focus on the use of antagonistic rhizobacterial strains belonging to the genus Pseudomonas or Bacillus . However, the development of biocontrol products based on isolates belonging to the Gram-negative genus Serratia is now gaining momentum. Serratia plymuthica is a ubiquitous bacterium that has been preferentially recovered from rhizospheres all over the world, both as a free-living and endophytic organism. Specific strains of S. plymuthica produce a broad palette of antimicrobial compounds and might hold great potential as broad-spectrum biocontrol agents. This review surveys the advances of biocontrol research with respect to plant-associated S. plymuthica strains focusing on the principles and mechanisms of action of S. plymuthica and their use or potential use for the biological control of fungal plant diseases. A cursory overview of the taxonomy and ecology of S. plymuthica is also provided. We highlight recent progress in the identification of antifungal secondary metabolites produced by S. plymuthica and pay special attention to the regulatory mechanisms underpinning the production of the latter metabolites. Finally, we discuss several strategies that may provide a basis to improve the efficacy of S. plymuthica -mediated biocontrol.

Proteolytic activity and antibiotic production by Trichoderma harzianum in relation to pathogenicity to insects

Two strains of Trichoderma harzianum, 101645, an insect pathogen and 206040, used for biological control of fungal plant pathogens were investigated for the production of serine protease, chitinase and antibiotic activity in relation to entomopathogenicity. Both strains produced serine protease with a M r of 31 kDa and chitinase with a M r of 44 kDa. Enzymes from both strains had similar characteristics and were produced during the growth phase. Both strains also produced peptaibols active against fungi in late growth and stationary phases which differed in their amino-alcohol content. The peptaibols were insecticidal when fed to larvae of Tenebrio molitor or when applied to the cuticle together with the serine protease. The results suggest that the virulence factors involved in biocontrol are the same as those for insect pathogenicity. This may affect the use of Trichoderma spp. for biocontrol as there may be effects on non-target insect species.

Extracellular Proteases ofTrichodermaSpecies

Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

Production of Extracellular Proteases by Human PathogenicTrichoderma longibrachiatumStrains

Species belonging to the filamentous fungal genus Trichoderma are well known as potential candidates for the biological control of plant pathogenic fungi and as cellulase producers of biotechnological importance. Several data were published in the last decade also about the clinical importance of this genus, indicating that Trichoderma strains may be potential opportunistic pathogens in immunocompromised patients. However, there is a lack of information about the potential virulence factors of clinical Trichoderma strains. This study was designed to examine the extracellular proteolytic enzymes of six clinical T. longibrachiatum isolates. Supernatants from induced liquid cultures of the examined strains were screened for proteolytic enzyme activities with 11 different chromogenic p-nitroaniline substrates. The production of trypsin-like, chymotrypsin-like and chymoelastase-like protease activities cleaving N-Benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide, N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, and N-Succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, respectively, was common among the strains examined. Separation of trypsin- and chymotrypsin-like activities by column chromatography revealed, that both systems are complex consisting of several isoenzymes. The pH-dependence of these two protease systems was also studied. Based on the results, the different isoenzymes seem to have different optimal pH values. Extracellular proteolytic enzymes may be involved in the pathogenecity of Trichoderma strains as facultative human pathogens.

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors α-difluoromethylarginine and α-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.

Inhibitory Effects of Super Reductive Water on Plant Pathogenic Fungi

The antifungal activity of super reductive water (SRW) against plant pathogenic fungi was examined to extend its application to integrated pest management (IPM) for plant diseases. Diluted solutions (<TEX>$\times$</TEX>1/10, <TEX>$\times$</TEX>1/25, and <TEX>$\times$</TEX>1/50) of SRW inhibited fungal growth of kiwifruit soft rot pathogen, Diaporthe actinidiae, in a concentration dependent manner, When kiwifruits were inoculated on wounds with mycelium blocks, stock and diluted solutions successfully inhibited the disease development. In addition to the high pH of the SRW, fungistatic activity was also considered as the cause of the antifungal effect against the pathogen. Whereas conidial germination of Magnaporthe grisea was not affected by the diluted SRW solutions, appressorium formation was significantly inhibited in a concentration dependent manner, With little harmfulness to human health and environment SRW could be used to control plant pathogenic fungi, particularly appressorium-forming fungal pathogens.

Monoclonal Antibodies Incorporated into Neotyphodium coenophialum Fungal Cultures: Inhibition of Fungal Growth and Stability of Antibodies

Three monoclonal antibodies (mAbs) produced against proteins from the tall fescue (Festuca arundinacea Schreb.) fungal endophyte Neotyphodium coenophialum hybridize exclusively to a fungal protein under denaturing conditions. The protein is approximately 88 kDa in size. These mAbs were individually incorporated into liquid medium to determine their effects on fungal growth in culture. Neotyphodium-specific mAbs inhibited fungal growth for the duration of the study. Fungal cultures grown in the presence of Neotyphodium-naive mAbs or in the absence of all mAbs grew unimpeded. Bright-field microscopy and immunohistochemical studies of cultures containing Neotyphodium-specific mAbs revealed a change in mycelia morphology with clumps exhibiting a gelatinous matrix containing sparse hyphae, while cultures receiving Neotyphodium-naive mAbs in medium demonstrated unrestricted growth with overlapping and branched hyphae. In liquid culture devoid of fungal isolates, mAbs were stable and detected throughout the experiment, but were below threshold detection levels within 15 min following inclusion in liquid cultures containing Neotyphodium spp., indicating rapid binding to fungal mycelia. Monoclonal antibodies may provide a new method to help control plant pathogenic fungi where chemical or genetic means are not feasible.

Effects of increased nitrate availability on the control of plant pathogenic fungi by the soil bacterium Bacillus subtilis

In wet soils, low oxygen conditions often develop that favour disease development by many soil-borne plant pathogens. The introduction of a biocontrol agent, to suppress disease development, would require that the agent remains metabolically active under such conditions. Denitrifying bacteria can maintain this metabolic activity by switching to nitrate respiration. In the rhizosphere, plant roots not only supply carbon as an electron donor, but also cause a localised lowering of oxygen concentrations, conditions favourable for nitrate respiration. Two strains of Bacillus subtilis, showing strong inhibition of a number of pathogenic fungi on agar plates, and the capacity to grow under anoxic and anaerobic conditions when provided with nitrate, were used to study the possible involvement of nitrate respiration in fungal disease control. The effect of the addition of nitrate on the activity of these antagonistic strains was studied under anoxic conditions using the sealed plate method of Fiddaman and Rossal [Fiddaman, P.J., Rossal, S., 1995. Plant Pathol. 44, 695–703]. The assay tests the activity, measured as a reduction in fungal growth, of antifungal volatiles (AFV) produced by the bacteria. The in vitro experiments showed that antagonism by the B. subtilis strains towards Fusarium oxysporum varied under anoxic conditions, depending on the nitrate availability and agar used as a growth medium. AFV activity was increased by the presence of nitrate in the medium at concentrations of 10 mM or more. Nitrate respiration may therefore have an important role in the control of fungal root diseases by allowing denitrifying soil-borne bacteria to remain metabolically active in wet soils with low oxygen concentrations.

Growth and polyamine metabolism inPyrenophora avenae exposed to cyclohexylamine and norspermidine

The effectiveness of inhibitors of polyamine biosynthesis in controlling plant pathogenic fungi is well established. The spermidine synthase inhibitor cyclohexylamine (CHA) and the spermidine analogue norspermidine were evaluated againstin vitro growth of the oat stripe pathogenPyrenophora avenae. Mycelial growth was reduced by 55% upon exposure to 2.0mM CHA while the same concentration of norspermidine reduced growth by 63%. Neither inhibitor had any effect on ODC or AdoMetDC activities, nor the flux of label from ornithine through to the polyamines. Levels of free polyamines in fungal tissue exposed to 0.01 mM norspermidine were unaltered, although 1.0mM CHA did produce a 75% increase in fungal putrescine content. These data suggest that CHA and norspermidine do not reduce fungal growth as a result of a perturbation in polyamine biosynthesis.

Fungicidal Activity of Two Spermidine Analogues

AbstractThe potential use of polyamine analogues as inhibitors of polyamine biosynthesis to control plant pathogenic fungi is well established. However, all of this information relates to the use of putrescine analogues and no data exist for spermidine analogues. In the present work, two spermidine analogues. N1‐ and N8‐acetylspermidine were evaluated against powdery mildew on barley. Post‐inoculation treatments reduced infection by 69.7% and 51.5%. respectively. Since the barley powdery mildew fungus cannot be grown axenically. mode of action studies were undertaken using the oat leaf‐stripe pathogen Pyrenophora avenae. Neither of the analogues had any effect on polyamine biosynthesis in P. avenae grown in vitro. Although the mechanism(s) by which inhibitors affect in vivo fungal growth and in vitro growth may differ, it is unlikely that the antifungal properties of the analogues are the result of a perturbation in polyamine biosynthesis.

Synergistic effects of a cellulase-producing Micromonospora carbonacea and an antibiotic-producing Streptomyces violascens on the suppression of Phytophthora cinnamomi root rot of Banksia grandis

Three polyvalent Streptomyces phages were used to isolate four Micromonospora species (M. carbonacea, M. chalcea, M. purpureochromogenes, and M. inositola) from mine-site rhizosphere soils in Western Australia. Streptomyces violascens was isolated using selective isolation techniques from the same soils. The Micromonspora spp. were examined for their ability to produce cellulases. Micromonospora carbonacea, M. chalcea, and M. purpureochromogenes, which were found to produce the enzyme, caused lysis of Phytophthora cinnamomi hyphae. Glasshouse trials showed that the use of the cellulase-producing M. carbonacea isolate, in conjunction with the antibiotic-producing S. violascens isolate, had a synergistic effect on the suppression of the Phytophthora root rot and in promoting growth of Banksia grandis. The importance of using a number of antagonists with different antagonistic abilities to control plant pathogenic fungi is discussed. Keywords: biological control, Micromonospora carbonacea, Streptomyces violascens, cellulases, Phytophthora cinnamomi.

Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum

There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene ( ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5′ terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the λgt11 library which contained the 5′ terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulons, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.

Wireworm Control on Potato, 1991

Abstract Certified LaRouge' seed pieces were planted on 19 Nov 1990 in a 0.4 acre area with Perrine marl soil and spaced 4 inches apart in rows that were 3 ft apart. Nine treatments were arranged in a randomized complete block design with 4 replications. Treatment plots consisted of 4 rows 12.2 m long. Plants were fertilized with 1700 lb/acre of granular fertilizer (6:12:10) at planting. Monitor was applied (1.0 qt [AI]/acre) on two dates and Pounce was applied (6 oz [AI]/acre) on four dates to control green peach aphid, Myzus persicae (Sulzer), and Thrips palmi Karny. In addition, Trigard was applied (1.2 lb [AI]/acre) on 17 Jan to control leafminers, Liriomyza sativae Blanchard and L. trifolii (Burgess). Topsin was applied (1 lb [AI]/acre) on 17 and 21 Jan to control plant pathogenic fungi. The insecticides tested were: EXP 60166A, Thimet, PCC300 (10% [AI] ethoprop and 10% [AI] phorate), and PCC 307 (8% [AI] fonofos and 12% [AI] phorate). Treatments evaluated were: EXP 60166A (0.1, 0.2, and 0.3 lb [AI]/acre), Thimet (3.6 lb [AI]/acre), PCC 300 (2.0 and 3.0 lb [AI]/acre), PCC 307 (1.9 and 2.5 lb [AI]/acre), and a nontreated check. Insecticides were manually sprinkled over the seed pieces in the furrow and covered with soil four days after planting. Vines were killed on 19 Feb 1991 with Paraquat Plus (1 qt/acre; 29.1% [AI]). Two 20 ft sections of row per treatment plot were harvested on 7 Apr 1991. Tubers were washed, graded for wireworm injury (tubers with ≤2 holes per tuber with a combined depth ≤3/4 inch) or damage (tubers with at least 1 to 2 holes per tuber with a combined depth &amp;gt;3/4 inch or &amp;gt;2 holes per tuber), cracking, sunburn, disease, etc., and fresh weight. Data for injury, damage, sunburn, and cracking are presented as a percentage of total fresh weight per harvested section of row.

Control of Plant Diseases by Chitinase Expressed from Cloned DNA inEscherichia coli

When Sclerotium rolfsii was sprayed with partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was found to be effective in reduction of disease incidence caused by S. rolfsii in beans and Rhizoctonia solani in cotton under greenhouse conditions (62% disease reduction in both diseases). A similar effect was obtained when we used viable cells of E. coli containing the plasmid pLCHIA. However, E. coli carrying the plasmid lacking the pL promotor did not have any effect. These results suggest a role for chitinase in biological control of plant pathogenic fungi