Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen-suppressive soil

Abstract

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF(103) of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Chitin Amendment Increases Soil Suppressiveness toward Plant Pathogens and Modulates the Actinobacterial and Oxalobacteraceal Communities in an Experimental Agricultural Field

“…From previous work based on isolated microbial community members, it has been postulated that the addition of chitin to soil stimulates bacterial communities more than fungal ones (8,9). Among the bacterial isolates obtained in several studies, members of the genus Streptomyces were dominant (10), followed by Stenotrophomonas and Bacillus (1,11,12). The presence of these bacterial chitinolytic groups was confirmed by analyses based on the 16S rRNA and chitinase (chiA) genes (11,13).…”

“…Among the bacterial isolates obtained in several studies, members of the genus Streptomyces were dominant (10), followed by Stenotrophomonas and Bacillus (1,11,12). The presence of these bacterial chitinolytic groups was confirmed by analyses based on the 16S rRNA and chitinase (chiA) genes (11,13). Experiments performed in plots of natural fields, in which chitin was placed in litter bags, have addressed and revealed the molecular diversity of the chitinases produced by Streptomyces (14).…”

“…Moreover, our understanding of the relationship of chitin and chitin degradation status to crop rotation and soil characteristics is also limited (13,15). As suggested from the above studies, members of the actinobacteria, which are ubiquitous in, and have been widely isolated from, agricultural soils, have been indicated as key degraders of complex organic molecules like chitin in the field (11,16). However, in experiments performed in microcosms under laboratory conditions, particular members of the Gamma-and Betaproteobacteria were found to become dominant after the addition of chitin (17).…”

The Millardetian Conjunction in the Modern World

“…This should help to reveal the huge potential of suppressive soils for managing soilborne pathogens. Characterization of the potential may be "easy" when biological nature of the suppression is known as illustrated by studies of soils with known chitinase and antifungal activities (Hjort et al, 2010). Metagenomics may also lead to screen uncultured microorganisms from soil which represent a potentially rich source of useful natural products.…”

From Dust Bowl to Dust Bowl: Soils are Still Very Much a Frontier of Science

“…Yet, the results to date are falling short of expectations. For instance, it remains unclear what percentage of the DNA present in soils is extracted by current techniques, and how representative this fraction is (Thakuria et al, 2008;Hjort et al, 2010;Trevors, 2009). This is clearly illustrated in a study by Ascher et al (2009), who applied a novel DNA extraction protocol to two forest soils and observed that sequential extraction leads to increased DNA recovery, that extracellular DNA can represent a very sizeable (34.6%) portion of the sum of extracellular and intracellular DNA, and that a very different perception of microbial diversity emerges if one considers the total extracted DNA or just the extracted intracellular DNA.…”