Identification of strains of Rhizobium leguminosarum

Abstract

Agriculture in New Zealand relies heavily on mixed pastoral crops. Legumes, such as white clover in particular, are extremely important due to their symbiotic relationship with Rhizobium spp. Many of these pastoral areas can be subject to dry conditions and drought. For Rhizobium spp. it is important to understand what effect desiccating stress can have and whether there is natural variation in the ability of individual strains to tolerate such stress. This could inform the development of better commercial inoculants and enable them to persist better in dry soils. Numerous researchers have investigated the nodulation ability and nitrogen fixation efficacy of R. leguminosarum bv. trifolii when in symbiosis with Trifolium repens (white clover). However, little research has focused on the ability of R. leguminosarum to live in soil as a saprophyte. This study aimed to investigate variation in desiccation tolerance of individual strains of R. leguminosarum bv. trifolli and to determine if it was possible to identify strains with higher tolerance to this stress. Twenty-four strains of R. leguminosarum were randomly selected from a collection obtained from sites that different in their annual soil moisture deficit (SMD). Twelve strains were chosen from three sites with high annual SMD (>100 days; dry sites) and 12 from three sites with low SMD (<5 days; wet sites). Sequencing of the 16S rRNA identified the strains as R. leguminosarum. Genotyping of the selected strains showed they were genetically diverse will 11 genetic groups identified, of which 6 were unique to a single strain. Two in vitro assays were used to identify strains that were tolerant of desiccation stress. The first assay measured relative biofilm (polysaccharide) production which has been linked to the ability to withstand desiccation stress in the literature. Following staining with crystal violet the results showed that strains produced significantly different quantities of biofilm (24 (P≤0.001), and 48 (P≤0.001) h incubation times) and this placed each strain into one of 8 different groups. The second assay investigated the ability of the strains to grow when placed under strong osmotic stress as osmotic stress involves a similar response pathway to desiccation stress in bacteria. The strains were incubated in two different concentrations of polyethylene glycol (PEG). There results showed there was a significant difference (P≤0.005) between the incubation times with more biofilm formed after 48 h than 24 h. There was also a significant difference in the amount of biofilm formed by individual isolates after 24 (P≤0.001), and 48 (P≤0.001) hours incubation, with the combined data placing the strains into 9 groups when grown in 50% PEG and 5 groups when grown in 60% PEG. The strain that produced the most biofilm was 53 which originated from a wet site and the strains growing the most when exposed to PEG were 34 (50% Peg) and 42 (60% PEG). To determine whether variation in genes producing the polysaccharide trehalose might explain variation in biofilm production or tolerance to PEG primers were developed to amplify five trehalose biosynthesis genes. These were trehalose-6-phosphate synthase (otsA), trehalose-6phosphate phosphatase (otsB), maltooligosyltrehalose synthase (treY), maltooligosyltrehalose trehalohydrolase (treZ) and trehalose synthase (treS). Only otsB produced readable DNA sequences suitable for analysis. When the DNA sequences were translated in silico there was a 98% identity between the amino acid sequences of the strains with six silent or conservative and two non-conservative substitutions. The two non-conservative substitutions were in isolates 32 and 42 and consisted of amino acid substitutions Serine to Alanine and Glutamic acid to Alanie. There was no obvious link between these amino acid substitutions and the ability of strains to produce more biofilm or grow in high concentrations of PEG. To measure the survival of strains in soil spontaneous antibiotic mutants were created that were tolerant of erythromycin. The survival of the strains with the most biofilm formation (53) and greatest growth under the highest PEG concentration (42) were compared to two average strains and the commercial strain TA1. The biofilm assay was initially used to determine that the ranking of isolates and the relative ability of mutant and wild type did not differ. However, the results were inconclusive. The results showed that after 4 and 45 day in dry soil held at room temperature there were significant differences between isolates. After 4 d incubation strain 42 had a greater number of CFUs than isolate 47, and after 45 days strain 53 had a greater number of CFU compared to strain 50 and TA1. For all strains there was the expected decrease in CFU over time. Overall this study identified that there is variation in the ability of strains of R. leguminosarum to withstand desiccation stress in soil. Strains 42 and 53 were selected using in vitro testing to be dry tolerant and survived better than other strains in dry soil. This indicated that the in vitro assays used here may be useful to rank strains of R. leguminosarum however, a greater number of strains, with more replication should be done to confirm this. This work highlights the potential of selecting strains to withstand desiccation and further development of a more robust screening process would assist desiccation tolerant commercial strains to be developed.