Thatch biodegradation and antifungal activities of two lignocellulolytic Streptomyces strains in laboratory cultures and in golf green turfgrass

The use of lignocellulolytic Streptomyces spp. as biological agents, to enhance thatch degradation in turf and to slow its rate of accumulation while controlling fungal growth in the thatch layer, was studied. In flask scale studies, two lignocellulolytic Streptomyces violaceusniger (= hygroscopicus) strains (YCED9 and WYE53) decomposed thatch (> 30% dry weight) over a 12-week incubation period. Biodegradation was accompanied by production of extracellular cellulases, xylanases, and peroxidases. The accumulation of the polymeric, water-soluble lignin degradation intermediate acid, precipitable polymeric lignin (APPL), was also observed. Residual thatch from 12-week-old cultures had an increased lignin-to-carbohydrate ratio, an indication that although lignin was metabolized, carbohydrates were preferential carbon sources for these actinomycetes. A spore-containing soluble dry powder formulation was used as an inoculum in an in situ field experiment. This formulation was maintained in storage at 4 degrees C for over two years without viability loss. Results from the golf green experiment showed that although treated thatch layers in established greens were not appreciably reduced over the course of one summer, the Streptomyces were active and maintained their populations within the thatch, while fungal growth was suppressed as compared to controls. The results show that treatment of turfgrass with these Streptomyces may be useful for the long-term control of fungal populations within the thatch. Longer field studies are required to assess the long-term potential for also controlling thatch build-up and fungal pathogens.     

In vitro and in vivo antagonism of Streptomyces violaceusniger YCED9 against fungal pathogens of turfgrass

The biocontrol agent Streptomyces violaceusniger YCED9 inhibited in vitro growth of seven fungal pathogens of turfgrass. Three different antibiotics were produced by the actinomycete, including nigericin, geldanamycin and a complex of macrocyclic lactone antibiotics. Each had a different spectrum of antifungal activity. Only nigericin was detected in soil or in grass rhizospheres inoculated with YCED9. However, all three antibiotics were produced when YCED9 was grown in a mixture of moistened grass and thatch. In greenhouse experiments, a grass seedling disease caused by the Rhizoctonia solani and a crown-foliar disease caused by Sclerotinia homeocarpa were partially controlled with commercial spore formulations of YCED9.     

Enhancement of Disease Control Efficacy of Chemical Fungicides Combined with Plant Resistance Inducer 2,3-Butanediol against Turfgrass Fungal Diseases

Turfgrass, the most widely grown ornamental crop, is severely affected by fungal pathogens including Sclerotinia homoeocarpa, Rhizoctonia solani, and Magnaporthe poae. At present, turfgrass fungal disease management predominantly relies on synthetic fungicide treatments. However, the extensive application of fungicides to the soil increases residual detection frequency, raising concerns for the environment and human health. The bacterial volatile compound, 2,3-butanediol (BDO), was found to induce plant resistance. In this study, we evaluated the disease control efficacy of a combination of stereoisomers of 2,3-BDO and commercial fungicides against turfgrass fungal diseases in both growth room and fields. In the growth room experiment, the combination of 0.9% 2R,3R-BDO (levo) soluble liquid (SL) formulation and 9% 2R,3S-BDO (meso) SL with half concentration of fungicides significantly increased the disease control efficacy against dollar spot and summer patch disease when compared to the half concentration of fungicide alone. In field experiments, the disease control efficiency of levo 0.9% and meso 9% SL, in combination with a fungicide, was confirmed against dollar spot and large patch disease. Additionally, the induction of defense-related genes involved in the salicylic acid and jasmonic acid/ethylene signaling pathways and reactive oxygen species detoxification-related genes under Clarireedia sp. infection was confirmed with levo 0.9% and meso 9% SL treatment in creeping bentgrass. Our findings suggest that 2,3-BDO isomer formulations can be combined with chemical fungicides as a new integrated tool to control Clarireedia sp. infection in turfgrass, thereby reducing the use of chemical fungicides.     

Antagonism ofPseudomonas cepacia against phytopathogenic fungi

Two strains ofPseudomonas cepacia, RJ3 and ATCC 52796, have been identified as potential antagonists of fungal plant pathogens. We have compared the antagnonistic activity of these two strains against various fungal pathogens. Although both strains displayed high levels of antagonism, ATCC 52796 was slightly more antagonistic than RJ3. The antagonist from RJ3 has been identified as the antifungal compound pyrrolnitrin after purification by HPLC and characterization by UV, IR, NMR, and mass spectroscopy. Both strains also antagonized the fungi by production of volatile compound(s), which have not yet been identified. Both strains are similar with respect to in vitro antagonism, mechanism of antagonism, and sensitivity to antibiotics.     

Pyrimethanil: Between efficient fungicide against Aspergillus rot on cherry tomato and cytotoxic agent on human cell lines

Pyrimethanil, a synthetic fungicide widely used for the treatment of pre‐ and postharvest fungal diseases on different agricultural crops, was explored for its antifungal activity on different fungal strains. The effect of pyrimethanil on fungal ergosterol was tested by using Aspergillus niger as a model organism. Furthermore, it was investigated, if pyrimethanil can effectively reduce the appearance of Aspergillus rot in wounded cherry tomato fruits. The fungicide cytotoxic effect on different human cell lines was evaluated. In addition, its influence on cell proliferation was studied. A. niger was the most resistant fungal strain (MFC 1.88 μg μL^?1) to the effect of pyrimethanil. Addition of ergosterol doubled the MFC on A. niger, indicating that the compound might interfere with ergosterol, main sterol of fungal cell membrane. Disease incidence of A. niger in wounded cherry tomato fruits was not detected with pyrimethanil treatment of 0.75 mg/wound. Some cytotoxic effects of pyrimethanil on human cell lines were recorded already at 50 ng μL^?1, while the expression of Ki67 marker of proliferation was decreased with 150 ng μL^?1. These results altogether indicate that pyrimethanil is effective in reducing various fungal pathogens, but further use of this fungicide should be reevaluated because of its cytotoxicity.     

Antagonistic Potential of Bacterial Species against Fungal Plant Pathogens (FPP) and Their Role in Plant Growth Promotion (PGP): A Review

Since the 19^th century to date, the fungal pathogens have been involved in causing devastating diseases in plants. All types of fungal pathogens have been observed in important agricultural crops that lead to significant pre and postharvest losses. The application of synthetic fungicide against the fungal plant pathogens (FPP) is a traditional management practice but at the same time these fungicides kill other beneficial microbes, insects, animal, and humans and are harmful to environment. The antagonistic microorganism such as bacteria are being used as an alternate strategy to control the FPP. These antagonistic species are cost-effective and eco-friendly in nature. These biocontrol bacteria have a broad mechanism against fungal pathogens present in the phyllosphere and rhizosphere of the plant. The antagonistic bacteria have different strategies against the FPP, by producing siderophore, biofilm, volatile organic compounds (VOCs), through parasitism, antibiosis, competition for limited resources and induce systemic resistance (ISR) in the host plant by activating the immune systems. The commercial bio-products synthesized by the major bacterial species Pseudomonas syringae, Burkholderia cepacia, Streptomyces griseoviridis, Pseudomonas fluorescens and Bacillus subtilis are used to control Fusarium, Pythium, Rhizoctonia, Penicillium, Alternaria, and Geotrichum. The commercial bio-formulations of bacteria act as both antifungal and plant growth regulators. The Plant growth-promoting rhizobacteria (PGPR) played a significant role in improving plant health by nitrogen-fixing, phosphorus solubilization, phytohormones production, minimizing soil metal contamination, and by ACC deaminase antifungal activities. Different articles are available on the specific antifungal activity of bacteria in plant diseases. Therefore, this review article has summarized the information on biocontrol activity of bacteria against the FPP and the role of PGPR in plant growth promotion. This review also provided a complete picture of scattered information regarding antifungal activities of bacteria and the role of PGPR.

     

Isolation and characterization of potent antifungal strains of the Streptomyces violaceusniger clade active against Candida albicans

Streptomyces strains were isolated from a sagebrush rhizosphere soil sample on humic acid vitamin (HV) agar and water yeast extract (WYE) agar supplemented with 1.5% (w/w) phenol as a selective medium. Acidic, neutral and alkaline pH conditions were also used in the isolation procedures. The phenol treatment reduced the numbers of both actinomycetes and non-actinomycetes on plates under all three pH conditions. From phenol-amended HV and WYE agar, 16 strains were isolated in pure culture; 14 from the HV agar and two from the WYE agar. All the isolates were tested for their antifungal activities against Pythium ultimum P8 and five yeast strains, including two antifungal drug-resistant Candida albicans strains. HV isolates that showed broad-spectrum antifungal antibiotic activities were all found to be members of the Streptomyces violaceusniger clade, while those that did not were non-clade members. The phenol treatment was not selective for S. violaceusniger clade members. Therefore, we tested the spores of both S. violaceusniger clade and non-clade members using two biocides, phenol and hydrogen peroxide, as selection agents. Spores of non-clade members, such as S. coelicolor M145 and S. lividans TK 21, survived these two biocides just as well as S. violaceusniger clade members. Thus, in our hands, biocide resistance was not S. violaceusniger clade specific as previously reported. However, isolates showing broad-spectrum antifungal and antiyeast activity were all members of the clade. We conclude that screening of isolates for broad-spectrum antifungal/antiyeast activity is the preferred method of isolating S. violaceusniger clade strains rather than biocide-based selection. Phylogenetic analysis of the phenol-resistant isolates revealed that the HV isolates that exhibited broad-spectrum antifungal antibiotic activity were all clustered and closely related to the S. violaceusniger clade, while the isolates that did not exhibit antifungal antibiotic activity were all non-clade members.     

撕裂蜡孔菌(Emmia lacerata)SR5抑菌特性及生防潜力评价

【背景】撕裂蜡孔菌(Emmia lacerata)是一种在世界范围内广泛分布的白腐真菌,对植物病原真菌有较好的抑制作用,可作为生防真菌进行开发和利用。【目的】对撕裂蜡孔菌SR5的抑菌能力和胞外产铁载体能力进行测定,挖掘其生防潜力。【方法】采用平板对峙法检测SR5对9种植物病原真菌的抑菌能力,并通过不同浓度的发酵原液测定真菌胞外代谢物的抑菌效果;结合铬天青S(chrome azurol S, CAS)检测法测定真菌产铁载体能力,明确SR5抑菌特性。【结果】SR5以过度生长的方式快速竞争营养和生存空间,拮抗9种植物病原真菌,抑菌率为23.7%–62.7%,对可可毛色二孢(Lasiodiplodia theobromae)的拮抗等级为Ⅳ级,而对其余8种病原真菌的拮抗等级为Ⅲ级,其中对香港丽赤壳(Calonectria hongkongensis)和间座壳(Diaporthe sp.)抑菌效果最佳;CAS检测法表明SR5能产生分泌型铁载体,产铁载体能力中等,最高铁载体活性单位(siderophore unit, SU)为44.1%。【结论】SR5以过度生长方式快速竞争营养和生存空间,而且以分泌...     

粮食上木霉菌的分离鉴定及其生防效果

【背景】粮食在生长和收储期极易受到病原真菌或产毒真菌的污染,造成严重的损失。众多实践证明木霉属(Trichoderma)可以有效防治植物病原真菌。【目的】鉴定和筛选能有效抑制粮食常见危害真菌的木霉生防菌株,开发生防菌剂,保障粮食生产安全。【方法】从粮食上分离筛选出35株木霉,通过多基因系统发育分析和形态学观察方法进行菌种鉴定,利用平板对峙试验筛选出对粮食常见危害真菌有抑制作用的菌株。【结果】35株木霉分属于8个种,分别为非洲哈茨木霉(Trichodermaafroharzianum)、类棘孢木霉(Trichodermaasperelloides)、 Trichoderma amoenum、近深绿木霉(Trichoderma paratroviride)、Trichoderma obovatum、长枝木霉(Trichoderma longibrachiatum)、东方木霉(Trichodermaorientale)和深绿木霉(Trichodermaatroviride)。对峙试验结果表明,这8种木霉对于粮食上分离到的10种危害真菌均具有较好的抑制效果。非洲哈茨木霉(T.afroharzi...     

Increased antifungal and chitinase specific activities of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> CECT 2413 by addition of a cellulose binding domain

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.     

Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond

Fungal infections are underestimated threats that affect over 1 billion people, and Candida spp., Cryptococcus spp., and Aspergillus spp. are the 3 most fatal fungi. The treatment of these infections is performed with a limited arsenal of antifungal drugs, and the class of the azoles is the most used. Although these drugs present low toxicity for the host, there is an emergence of therapeutic failure due to azole resistance. Drug resistance normally develops in patients undergoing azole long-term therapy, when the fungus in contact with the drug can adapt and survive. Conversely, several reports have been showing that resistant isolates are also recovered from patients with no prior history of azole therapy, suggesting that other routes might be driving antifungal resistance. Intriguingly, antifungal resistance also happens in the environment since resistant strains have been isolated from plant materials, soil, decomposing matter, and compost, where important human fungal pathogens live. As the resistant fungi can be isolated from the environment, in places where agrochemicals are extensively used in agriculture and wood industry, the hypothesis that fungicides could be driving and selecting resistance mechanism in nature, before the contact of the fungus with the host, has gained more attention. The effects of fungicide exposure on fungal resistance have been extensively studied in Aspergillus fumigatus and less investigated in other human fungal pathogens. Here, we discuss not only classic and recent studies showing that environmental azole exposure selects cross-resistance to medical azoles in A. fumigatus, but also how this phenomenon affects Candida and Cryptococcus, other 2 important human fungal pathogens found in the environment. We also examine data showing that fungicide exposure can select relevant changes in the morphophysiology and virulence of those pathogens, suggesting that its effect goes beyond the cross-resistance.     

Expression of a novel antimicrobial peptide Penaeidin4-1 in creeping bentgrass (Agrostis stolonifera L.) enhances plant fungal disease resistance

Background

Turfgrass species are agriculturally and economically important perennial crops. Turfgrass species are highly susceptible to a wide range of fungal pathogens. Dollar spot and brown patch, two important diseases caused by fungal pathogens Sclerotinia homoecarpa and Rhizoctonia solani, respectively, are among the most severe turfgrass diseases. Currently, turf fungal disease control mainly relies on fungicide treatments, which raises many concerns for human health and the environment. Antimicrobial peptides found in various organisms play an important role in innate immune response.

Methodology/Principal Findings

The antimicrobial peptide - Penaeidin4-1 (Pen4-1) from the shrimp, Litopenaeus setiferus has been reported to possess in vitro antifungal and antibacterial activities against various economically important fungal and bacterial pathogens. In this study, we have studied the feasibility of using this novel peptide for engineering enhanced disease resistance into creeping bentgrass plants (Agrostis stolonifera L., cv. Penn A-4). Two DNA constructs were prepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to the Pen4-1 coding sequence. A maize ubiquitin promoter was used in both constructs to drive gene expression. Transgenic turfgrass plants containing different DNA constructs were generated by Agrobacterium-mediated transformation and analyzed for transgene insertion and expression. In replicated in vitro and in vivo experiments under controlled environments, transgenic plants exhibited significantly enhanced resistance to dollar spot and brown patch, the two major fungal diseases in turfgrass. The targeting of Pen4-1 to endoplasmic reticulum by the transit peptide of AP24 protein did not significantly impact disease resistance in transgenic plants.

Conclusion/Significance

Our results demonstrate the effectiveness of Pen4-1 in a perennial species against fungal pathogens and suggest a potential strategy for engineering broad-spectrum fungal disease resistance in crop species.     

Phenotypic and biological properties of two antagonist <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains

Bacillus subtilis KB-1111 and KB-1122 were studied to illustrate their phenotypic and biological properties. Comparison of KB-1111 with KB-1122 in morphology was carried out by microscopy and agar plate assays. Biological assay of the test strains showed that they may possess different physiological pathways from those of reference strain ATCC6501. The assessment of antagonism against the indicator fungi showed that both test strains had broad antifungal characteristics against eight phytopathogenic fungi. Of those fungal species, Magnaporthe grisea P131, Sclerotinia sclerotiorum, and F. oxysporium exhibited high sensitivity to the test strains.     

Evaluation of Streptomyces for Biocontrol of Bipolaris sorokiniana and Sclerotinia homoeocarpa on the Phylloplane of Poa pratensis1

Several species of Streptomyces were evaluated for their ability to control Sclerotinia homoeocarpa (dollar spot) and Bipolaris sorokiniana (leaf spot) on the phylloplane of Poa pratensis (Kentucky bluegrass). Species evaluated included S. diastaticus (S32), S. galbus (S35), and S. hygroscopicus (isolates S13, S28). All evaluations were conducted on the upper epidermis of intact attached leaves of P. pratensis, and antagonism was measured as the ability of Streptomyces isolates to prevent chlorophyll loss from leaves inoculated with B. sorokiniana or S. homoeocarpa during pathogenesis. Only S. hygroscopicus (S13) effectively prevented infection and subsequent chlorophyll loss from leaves inoculated with B. sorokiniana or S. homoeocarpa. Isolate S28 of S. hygroscopicus showed erratic antagonism of both pathogens, depending upon how the isolate was prepared for use. Streptomyces diastaticus and S. galbus were antagonistic to S. homoeocarpa only in whole culture form.     

Characterization of <Emphasis Type="Italic">Streptomyces</Emphasis> spp. isolated from the rhizosphere of oil palm and evaluation of their ability to suppress basal stem rot disease in oil palm seedlings when applied as powder formulations in a glasshouse trial

Ganoderma boninense, the main causal agent of oil palm (Elaeis guineensis) basal stem rot (BSR), severely reduces oil palm yields around the world. To reduce reliance on fungicide applications to control BSR, we are investigating the efficacy of alternative control methods, such as the application of biological control agents. In this study, we used four Streptomyces-like actinomycetes (isolates AGA43, AGA48, AGA347 and AGA506) that had been isolated from the oil palm rhizosphere and screened for antagonism towards G. boninense in a previous study. The aim of this study was to characterize these four isolates and then to assess their ability to suppress BSR in oil palm seedlings when applied individually to the soil in a vermiculite powder formulation. Analysis of partial 16S rRNA gene sequences (512 bp) revealed that the isolates exhibited a very high level of sequence similarity (>?98%) with GenBank reference sequences. Isolates AGA347 and AGA506 showed 99% similarity with Streptomyces hygroscopicus subsp. hygroscopicus and Streptomyces ahygroscopicus, respectively. Isolates AGA43 and AGA48 also belonged to the Streptomyces genus. The most effective formulation, AGA347, reduced BSR in seedlings by 73.1%. Formulations using the known antifungal producer Streptomyces noursei, AGA043, AGA048 or AGA506 reduced BSR by 47.4, 30.1, 54.8 and 44.1%, respectively. This glasshouse trial indicates that these Streptomyces spp. show promise as potential biological control agents against Ganoderma in oil palm. Further investigations are needed to determine the mechanism of antagonism and to increase the shelf life of Streptomyces formulations.     

Transgenic creeping bentgrass with delayed dollar spot symptoms

Creeping bentgrass (Agrostis palustris Huds) is animportant turfgrass used on golf course greens and fairways. It is susceptibleto a number of fungal pathogens and requires considerable fungicide use fordisease control. Transgenic approaches may be useful in improving the level ofdisease resistance. We have generated transgenic creeping bentgrass plantsexpressing PR5K from Arabidopsis thaliana (L.) Henyh. PR5Kis a receptor protein kinase whose extracellular domain is homologous to thePR5family of pathogenesis-related proteins. In a field test of plants inoculatedwith the fungal pathogen dollar spot (Sclerotiniahomoeocarpa F.T. Bennett) four of the eight transgenic lines showeddelays in disease expression of 29 to 45 days, relative to the control plants.     

Mycolytic effect of fluorescent Pseudomonas in biocontrolling of fungal phytopathogenic Curvularia lunata,Fusarium oxysporum,Alternaria padwickii and Rhizoctonia solani

About 50 bacterial strains, each of Pseudomonas fluorescens, from different rhizospheric soil of different plants were screened for antagonistic activity against Curvularia lunata, Fusarium oxysporum, Alternaria padwickii, Rhizoctonia solani causing black kernel, kernel spotting, root rots, stackburn and sheath blight diseases of rice (Oryza sativa L.). Out of the 50 isolates, 15 isolates were found to be effective in lysing the cell wall of the above-mentioned putative pathogens tested in vitro. These Pseudomonas isolates produced mycolytic enzymes, viz. β-1,3-glucanases, β-1,4-glucanases and lipases. P. fluorescens PAK1 and PAK12 among the strains were more effective for the production of these enzymes while PAK12 produce good level of β-1,3-glucanases, β-1,4-glucanases and lipases against tested fungal pathogens. These findings demonstrate a mechanism of antagonism by P. fluorescens against different fungal plant pathogens.     

Functional characterization of the recombinant antimicrobial peptide Trx-<Emphasis Type="Italic">Ace</Emphasis>-AMP1 and its application on the control of tomato early blight disease

Ace-AMP1 is a potent antifungal peptide found in onion (Allium cepa) seeds with sequence similarity to plant lipid transfer proteins. Transgenic plants over-expressing Ace-AMP1 gene have enhanced disease resistance to some fungal pathogens. However, mass production in heterologous systems and in vitro application of this peptide have not been reported. In this study, Ace-AMP1 was highly expressed in a prokaryotic Escherichia coli system as a fusion protein. The purified protein inhibited the growth of many plant fungal pathogens, especially Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, and Verticillium dahliae. The inhibitory effect was accompanied by hyphal hyperbranching for V. dahliae but not for F. oxysporum f. sp. vasinfectum and A. solani, suggesting that the mode of action of Ace-AMP1 on different fungi might be different. Application of Ace-AMP1 on tomato leaves showed that the recombinant protein conferred strong resistance to the tomato pathogen A. solani and could be used as an effective fungicide.     

Synthesis and antifungal activity of imidazo[1,2-b]pyridazine derivatives against phytopathogenic fungi

A series of 3,6-disubstituted imidazo[1,2-b]pyridazine derivatives have been synthesized and characterized with spectroscopic analyses. The antifungal activities of these compounds against nine phytopathogenic fungi were evaluated by the mycelium growth rate method. The in vitro antifungal bioassays indicated that most of compounds displayed excellent and broad-spectrum antifungal activities. Especially, compounds 4a, 4c, 4d, 4l and 4r exhibited 1.9–25.5 fold more potent than the commercially available fungicide hymexazol against Corn Curvalaria Leaf Spot (CL), Alternaria alternate (AA), Pyricularia oryzae (PO) and Alternaria brassicae (AB) strains. Structure-activity relationship analysis showed that the enhanced antifungal activity is significantly affected by the substituents on the benzene ring and pyridazine ring.