Disease Suppressive Composts Research Articles

Characterization of Arthropeptide B, an Antifungal Cyclic Tetrapeptide from Arthrobacter humicola.

Disease suppressive composts are known, yet little information on the potential role of specific microbial antagonist within are available. Arthrobacter humicola isolate M9-1A has been obtained from a compost prepared from marine residues and peat moss. The bacterium is a non-filamentous actinomycete with antagonistic activity against plant pathogenic fungi and oomycetes sharing its ecological niche in agri-food microecosystems. Our objective was to identify and characterize compounds with antifungal activity produced by A. humicola M9-1A. Arthrobacter humicola culture filtrates were tested for antifungal activity in vitro and in vivo and a bioassay-guided approach was used to identify potential chemical determinants of its observed activity against molds. The filtrates reduced the development of lesions of Alternaria rot on tomatoes and the ethyl acetate extract inhibited growth of Alternaria alternata. A compound, arthropeptide B [cyclo-(L-Leu, L-Phe, L-Ala, L-Tyr)], was purified from the ethyl acetate extract of the bacterium. Arthropeptide B is a new chemical structure reported for the first time and has shown antifungal activity against A. alternata spore germination and mycelial growth.

Arthropeptide A, an antifungal cyclic tetrapeptide from Arthrobacter psychrophenolicus isolated from disease suppressive compost

In an effort to describe bioactive antifungal compounds from antagonistic bacteria with potential for biocontrol of plant pathogens, a strain of Arthrobacter psychrophenolicus was collected from plant disease suppressive compost prepared from composted material of marine origin. Few natural products have been characterized from the non-filamentous Actinobacteria genus Arthrobacter. A new cyclic tetrapeptide, cyclo-(L-Pro-L-Leu-L-γHyp-L-Tyr); arthropeptide A (1), was isolated from the EtOAc soluble culture filtrate extract of A. psychrophenolicus M9-17 grown in MOLP broth. Its structure was confirmed by HRMS, interpretation of NMR data, and a modified Marfey’s method. Arthropeptide A (1) displayed antifungal activity towards Alternaria alternata, the causal agent of disease in numerous host plant species, which had shown the previous susceptibility to A. psychrophenolicus. The newly identified compound may be responsible, in part, for the inhibitory activity of the bacterium against fungal plant pathogens.

Suppression of Fusarium dry rot of potato using beneficial bacterial treatments

Fusarium dry rot affects up to 60% of potato tubers during storage. Pathogen resistance development to synthetic chemical fungicides is a main contributing factor. Previous work has demonstrated the use of bacterial antagonists against plant pathogens. In this study, bacteria isolated from disease suppressive composts were tested to evaluate their antagonistic activity against F. sambucinum in vitro, as well as to assess their ability to reduce Fusarium dry rot of potato tubers. To identify the potential implication of antifungal compounds in their inhibitory activity, bacterial extracts were produced and assayed against F. sambucinum mycelial growth and conidial germination. In vitro results showed that all tested bacteria suppressed F. sambucinum mycelial growth over six days of incubation. When testing the bacteria against potato dry rot, 30 of 32 bacteria reduced the symptoms of the disease at the higher bacterial concentration tested. The combined in vitro and in vivo studies indicated Bacillus subtilis B9–8, Pseudomonas moraviensis F9–6, Pseudomonas koreensis F9–9, and Pseudomonas gessardii M9–16 were the most effective strains. Extracts from these four bacteria were able to inhibit mycelial growth and/or conidial germination of the fungal mold. Results from this study suggests that multiple antagonistic bacteria may be useful in controlling Fusarium dry rot on potato tubers. Extracts from the four most effective bacteria indicated that antibiosis was a main mode of action against F. sambucinum.

Harnessing the Microbiomes of Suppressive Composts for Plant Protection: From Metagenomes to Beneficial Microorganisms and Reliable Diagnostics.

Soil-borne diseases cause significant yield losses worldwide, are difficult to treat and often only limited options for disease management are available. It has long been known that compost amendments, which are routinely applied in organic and integrated farming as a part of good agricultural practice to close nutrient cycles, can convey a protective effect. Yet, the targeted use of composts against soil-borne diseases is hampered by the unpredictability of the efficacy. Several studies have identified and/or isolated beneficial microorganisms (i.e., bacteria, oomycetes, and fungi) from disease suppressive composts capable of suppressing pathogens (e.g., Pythium and Fusarium) in various crops (e.g., tomato, lettuce, and cucumber), and some of them have been developed into commercial products. Yet, there is growing evidence that synthetic or complex microbial consortia can be more effective in controlling diseases than single strains, but the underlying molecular mechanisms are poorly understood. Currently, a major bottleneck concerns the lack of functional assays to identify the most potent beneficial microorganisms and/or key microbial consortia from complex soil and compost microbiomes, which can harbor tens of thousands of species. This focused review describes microorganisms, which have been isolated from, amended to or found to be abundant in disease-suppressive composts and for which a beneficial effect has been documented. We point out opportunities to increasingly harness compost microbiomes for plant protection through an integrated systems approach that combines the power of functional assays to isolate biocontrol and plant growth promoting strains and further prioritize them, with functional genomics approaches that have been successfully applied in other fields of microbiome research. These include detailed metagenomics studies (i.e., amplicon and shotgun sequencing) to achieve a better understanding of the complex system compost and to identify members of taxa enriched in suppressive composts. Whole-genome sequencing and complete assembly of key isolates and their subsequent functional profiling can elucidate the mechanisms of action of biocontrol strains. Integrating the benefits of these approaches will bring the long-term goals of employing microorganisms for a sustainable control of plant pathogens and developing reliable diagnostic assays to assess the suppressiveness of composts within reach.

Compost bacteria provide antifungal activity against grey mold andAlternariarot on bell pepper fruit

Bell pepper (Capsicum annuum L.) fruit are susceptible to various molds, including grey mold and Alternaria rot. In this work, antagonistic bacteria isolated from disease-suppressive composts were assessed for their ability to reduce the mycelial growth of Botrytis cinerea Persoon and Alternaria alternata (Fries) Keissler as well as to suppress grey mold and Alternaria rot on bell pepper. Results showed that different bacterial antagonists reduced mycelial growth of B. cinerea and A. alternata by up to 49% and 53%, respectively. The bacteria provided inhibition of grey mold, reducing incidence and severity by as much as 43% and 67%, respectively. The isolates also provided strong inhibitory effects against Alternaria rot, decreasing incidence and severity by as much as 77% and 80%, respectively. Pseudomonas and Arthrobacter spp. were particularly suppressive to Alternaria rot. Results of this study suggest antagonistic bacteria may represent a potential method for controlling fruit rot of bell pepper.

Development of PCR-based molecular marker for screening of disease-suppressive composts against Fusarium wilt of tomato (Solanum lycopersicum L.).

The present study was carried out to develop a PCR-based molecular marker suitable for screening of disease-suppressive composts against Fusarium wilt of tomato. An effective uncultured bacterial community was screened from our previous study on investigation of microbial communities in composts for their potential for biocontrol of Fusarium wilt. Based on available sequence information (Accession no. HQ388491) of selective community, PCR-based molecular markers were designed and tested for their specificity in different compost sample. To confirm specificity of designed marker, real-time reverse transcription-PCR (qRT-PCR) analysis was performed. Selective marker efficacy was further tested for different set of composts and results were cross-verified by conducting bioassay of same composts against Fusarium wilt in tomato crop. Results showed that out of two designed set of primers (i.e., PAC1F/PAC1R and PAC4F/PAC4R), primer set PAC4F/PAC4R resulted in successful amplification of 199bp in highly disease-suppressive compost (i.e., CPP); however, no/below detection level amplification was observed in non-suppressive compost (JC). qRT-PCR analysis confirmed the specificity of selective marker by representing single peak in melting curve. A clear difference was observed in relative population of selective community in different set of composts. It was observed maximum in the most effective compost, i.e., CPP followed by other disease-suppressive composts. Cross-examination of results with bioassay confirmed that composts with presence of selective bacterial community having no/very less disease incidence of Fusarium. It is clearly evident from the study that such kind of molecular markers can be developed and used in future research focusing on compost-based disease suppression.

Desirable Traits of a Good Biocontrol Agent against Verticillium Wilt.

The soil-borne fungus Verticillium causes serious vascular disease in a wide variety of annual crops and woody perennials. Verticillium wilt is notoriously difficult to control by conventional methods, so there is great potential for biocontrol to manage this disease. In this study we aimed to review the research about Verticillium biocontrol to get a better understanding of characteristics that are desirable in a biocontrol agent (BCA) against Verticillium wilt. We only considered studies in which the BCAs were tested on plants. Most biocontrol studies were focused on plants of the Solanaceae, Malvaceae, and Brassicaceae and within these families eggplant, cotton, and oilseed rape were the most studied crops. The list of bacterial BCAs with potential against Verticillium was dominated by endophytic Bacillus and Pseudomonas isolates, while non-pathogenic xylem-colonizing Verticillium and Fusarium isolates topped the fungal list. Predominant modes of action involved in biocontrol were inhibition of primary inoculum germination, plant growth promotion, competition and induced resistance. Many BCAs showed in vitro antibiosis and mycoparasitism but these traits were not correlated with activity in vivo and there is no evidence that they play a role in planta. Good BCAs were obtained from soils suppressive to Verticillium wilt, disease suppressive composts, and healthy plants in infested fields. Desirable characteristics in a BCA against Verticillium are the ability to (1) affect the survival or germination of microsclerotia, (2) colonize the xylem and/or cortex and compete with the pathogen for nutrients and/or space, (3) induce resistance responses in the plant and/or (4) promote plant growth. Potential BCAs should be screened in conditions that resemble the field situation to increase the chance of successful use in practice. Furthermore, issues such as large scale production, formulation, preservation conditions, shelf life, and application methods should be considered early in the process of selecting BCAs against Verticillium.

Biofumigation with Fresh Manure or Brassicaceae Residuals could be a Promising Methyl Bromide Alternative in Head Lettuce Production

The current study investigates the natural alternatives of methyl bromide on head lettuce plants grown in greenhouse. Lettuce (Lactuca sativa L.) seedlings (cv. “Big Bell”) were treated with six different MBr alternatives which are, Biofumigation with fresh cow manure, fresh chicken manure, or Brassicaceae residuals plus Bio-Compost application (using disease suppressive compost) in comparison with chemical control with one of MBr chemical alternatives (Basamid) and finally negative control (which is the standard farmer treatment). Despite that chemical control with Basamid recorded the highest survival rate, biofumigation with fresh chicken manure gave the best head length, diameter, fresh weight, dry weight, total and marketable yield. Also, highest head quality characteristics such as TSS, SPAD reading, and nitrogen content were recorded with biofumigation with fresh chicken manure. Regardless of the superiority of Biofumigation with fresh chicken manure, Biofumigation with fresh cow manure or Brassicaceae residuals showed a better vegetative growth and quality parameters than farmer treatment (control), and they had significantly less effect than chemical control treatment. Bio-Compost fortified with Trichoderma harzianum and Bacillus subtilis (disease suppressive compost) recorded survival rate similar to biofumigation treatments but were significantly lower than chemical control treatment. Bio-Compost treatment did not result in a good result regarding total and marketable yield head fresh and dry weight. There was no detected significant effect of the studied treatments on stem diameter, L‑ascorbic acid (vitamin C), phosphorus and potassium content

Physiological and molecular characterization of compost bacteria antagonistic to soil-borne plant pathogens.

Disease suppressive composts have the potential to mitigate the risks associated with chemical pesticides. One of the main characteristics responsible for the suppressive nature of composts is their microbiological populations. To gain insight into the determinants responsible for their suppressive effects, we assayed composts to (i) isolate and identify beneficial antagonistic bacteria, (ii) quantify their antifungal and anti-oomycetal activities, (iii) extract inhibitory compounds produced by the bacteria, and (iv) identify antimicrobial lipopeptides produced by these bacteria. The antagonistic bacteria belonged to the genera Arthrobacter, Pseudomonas, Bacillus, Brevibacillus, Paenibacillus, and Rummeliibacillus and had the ability to antagonise the growth of Fusarium sambucinum, Verticillium dahliae, and (or) Pythium sulcatum. These bacteria produced antimicrobial compounds that affected the mycelial growth and (or) conidial germination of the pathogens. Mass spectrometry analyses showed the presence of various antimicrobial lipopeptides in Bacillus and Bacillus-related spp. extracts, demonstrating that they are responsible, at least in part, for the antagonistic activity of the bacteria. Results from this work provide greater insight into some of the biological, biochemical, and physiological determinants of suppressiveness in composts involved in the control of plant pathogens.

Microbial Diversity and Bioactive Substances in Disease Suppressive Composts from India

ABSTRACTThe present study aimed to investigate microbial communities in seven Indian composts and their potential for biocontrol of Fusarium oxysporum f. sp. lycopersici. In addition, identification of bioactive substances in disease suppressive composts was also attempted. Composts were chosen based on disease suppressiveness and subjected to molecular microbial analyses. Total genomic DNA from the composts was extracted and amplified with polymerase chain reaction using primers targeting the 18S rRNA and 16S rRNA genes of fungi and bacteria, respectively. Denaturing gradient gel electrophoresis (DGGE) fingerprinting and DNA sequencing were used to identify the fungal and bacterial targets. Phylogenetic analysis of the fungal 18S rRNA ITS gene sequences showed that phylum Ascomycota was dominant in all composts, while in the bacterial 16S rRNA gene sequences, the phylum Proteobacteria was dominant. Some fungi in disease suppressive composts grouped phylogenetically close to F. oxysporum. Bacterial sequences with close similarity (>95% identity) with Actinobacterium showed a strong presence only in disease suppressive composts. Disease suppressive composts formed a separate group in the cluster analysis of 18S rRNA ITS and 16S rRNA gene sequences. Gas chromatography-time of flight-mass spectrometry was performed with compost extracts to determine if bioactive substances were present in disease suppressive composts. The analysis of compost organic matter showed a negative association of disease suppressiveness with phloroglucinol, sitosterol, and monoenoic fatty acid, while cholesterol and certain organic acids were positively associated with suppressiveness.

Cytological and other aspects of pathogenesis-related gene expression in tomato plants grown on a suppressive compost.

Recent studies have shown that certain composts may trigger indirect defence mechanisms by sensitizing the plant to create an increased state of resistance, similar to systemic acquired resistance. In this study, the capacity of a disease-suppressive compost to alter the expression pattern of certain pathogenesis-related (PR) genes in the root system of tomato plants (Solanum lycopersicum) provided the opportunity to study their cellular expression pattern and to investigate putative roles of these genes in the mechanisms of plant defence. Employing the reverse transcription-polymerase chain reaction (RT-PCR) and in situ RNA:RNA hybridization techniques, the accumulation and distribution of the transcripts of the differentially expressed PR genes were examined in plants grown on compost and compared with those of control plants grown on peat. Elevated levels of expression of the pathogenesis-related genes PR-1, PR-5 and P69/PR-7 were detected in the roots of tomato plants grown on the compost. A clearly distinguished spatial induction pattern was observed for these PR genes: PR-1 transcripts were almost exclusively detected in the pericycle cells surrounding the root stele of the main and lateral roots; PR-5 transcripts were present in the phloem of the root and stem tissues; and the accumulation and distribution of PR-7 transcripts was detected in discrete groups of cells that appeared sporadically in both the parenchyma and vascular system of the root, suggesting that the gene is not expressed in a tissue-specific manner. In addition, a novel cDNA clone was isolated (P69G), which probably encodes a new tomato P69 isoform. This study provides evidence that a suppressive compost is able to elicit consistent and increased expression of certain PR genes in the roots of tomato plants, even in the absence of any pathogen. The in situ localization studies reveal expression patterns which are in accordance with the presence of protein or with the putative roles of the respective encoded proteins. The expression of the PR genes may be triggered by the microflora of the compost or could be associated with abiotic factors of the compost.

Inoculating Composted Pine Bark with Beneficial Organisms to Make a Disease Suppressive Compost for Container Production in Mexican Forest Nurseries

Abstract At a Mexican nursery, we compost pine bark, inoculate it with beneficial microorganisms, and use it to grow conifer seedlings in containers for reforestation. In this article, I describe how we make composted pine bark (CPB), a technically and economically viable alternative to expensive, imported growth substrates. CPB is easy to produce and can be made on a small scale, generating permanent nursery and reforestation jobs and promoting the stability of Mexican forest plantation companies. We have found that using CPB minimizes use of fungicides in the nursery. I discuss how to inoculate composted bark with bacteria (Bacillus spp.) and fungi (Trichoderma spp.) to enhance suppression of root disease organisms.

Inoculating Composted Pine Bark with Beneficial Organisms to Make a Disease Suppressive Compost for Container Production in Mexican Forest Nurseries

Abstract At a Mexican nursery, we compost pine bark, inoculate it with beneficial microorganisms, and use it to grow conifer seedlings in containers for reforestation. In this article, I describe how we make composted pine bark (CPB), a technically and economically viable alternative to expensive, imported growth substrates. CPB is easy to produce and can be made on a small scale, generating permanent nursery and reforestation jobs and promoting the stability of Mexican forest plantation companies. We have found that using CPB minimizes use of fungicides in the nursery. I discuss how to inoculate composted bark with bacteria (Bacillus spp.) and fungi (Trichoderma spp.) to enhance suppression of root disease organisms.

A new operation for producing disease-suppressive compost from grass clippings

This study evaluated the use of grass clippings discharged from golf courses as the raw material for production of a suppressive compost to control Rhizoctonia large-patch disease in mascarene grass. Bacillus subtilis N4, a mesophilic bacterium with suppressive effects on the pathogenic fungus Rhizoctonia solani AG2-2, was used as an inoculum in a procedure developed with the aim of controlling composting temperatures and inoculation timing. The population density of mesophilic bacteria in the raw material was reduced to around 5 log10 CFU/g (dry weight) of composting material in the self-heating reaction at the initial stage of composting by maintaining a temperature of 80 degreesC for 1 day. The inoculum was applied immediately, and the composting material was maintained at 40 degreesC for 3 days. This served both to highly concentrate the suppressive bacterium and to achieve sporulation. The temperature was then raised to 60 degreesC and maintained, enabling hygienic, high-speed composting while maintaining the population density of the suppressive bacterium as high as 8 log10 CFU/g (dry weight) in the compost. The suppressiveness of compost made in this way was confirmed in a turf grass disease prevention assay.