Effects of endophytic actinobacteria on lucerne growth and the development of its N2-fixation symbiosis with rhizobia

Author: Hoang Xuyen Le

Le, Hoang Xuyen, 2015 Effects of endophytic actinobacteria on lucerne growth and the development of its N2-fixation symbiosis with rhizobia, Flinders University, School of Medicine

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Lucerne (Medicago sativa L.) is grown in Australia for hay and livestock production. Improving the root health of lucerne and nitrogen fixation capacity has the potential to improve its establishment, growth and persistence. Endophytic actinobacteria which colonise plant roots have been reported to increase the growth and nodulation of legumes. The main aims of this study were to isolate endophytic actinobacteria from the healthy roots of different legumes (lucerne, pea, clover and medic) and screen the well-sporulating actinobacteria for their effects on the growth and nodulation of lucerne. The antifungal and bio-control activity of endophytic actinobacteria against Rhizoctonia solani and Pythium irregulare which can cause root rot of lucerne roots were also examined.

Two hundred and twenty five endophytic actinobacteria were successfully recovered from roots and nodules of the four legumes. Streptomyces (56%) was the dominant genus amongst the isolates; humic acid vitamin B and tap water yeast extract were the most effective media for isolation, and at least half the isolates were obtained from plates incubated at 37oC.

The endophytic actinobacteria had positive effects on lucerne seeds germinated on agar plates and in a sandy loam soil. Forty nine of the 148 well-sporulating isolates (33%) enhanced the germination of lucerne in terms of the number of viable seedlings and improved root length on agar. These cultures appear to show host bias as 33 of the 49 were isolated from lucerne. In a sterile sandy loam soil, 22 isolates improved both germination and root length when co-inoculated with rhizobia strain RRI 128. Twelve actinobacteria which improved germination and early lucerne growth were progressed to studies investigating their effect on lucerne growth, nodulation and nitrogen fixation in a series of pot experiments. As a result, two endophytic Streptomyces LuP30 and LuP47B were selected for further investigation as they increased the shoot weight by 87% and 88%, respectively, in a sandy loam medium. Shoot weight was increased by 25% and 35%, and the combined shoot and root weight by 26% and 30% by co-inoculation of S. meliloti RRI 128 with either LuP30 or LuP47B, respectively, in sand and vermiculite.

Streptomyces LuP30 and LuP47B were studied for their effects on lucerne growth and symbiosis at three different levels of nitrogen (3 mg, 25 mg and 50 mg NH4NO3 per kg soil). The actinobacteria had some effect independent of the rhizobial partner, as LuP30 increased the shoot dry weight of lucerne by 25% at 50 mg NH4NO3 while LuP47B increased the shoot dry weight by 30% and 23% at 25 mg and 50 mg NH4NO3, respectively, when applied the absence of rhizobia. Co-inoculation of either LuP30 or LuP47B with Sinorhizobium meliloti RRI 128 at 25 mg NH4NO3 per kg soil showed the greatest increases in plant growth, nodulation and symbiosis of lucerne. For example, co-inoculation of LuP30 with S. meliloti RRI 128 produced the largest increase in shoot weight of 46% with 25 mg NH4NO3. Co-inoculation with either LuP30 or LuP47B with the rhizobia increased the number of nodules by more than 100% compared with S. meliloti RRI128 alone after 4 weeks. In a labelled 15N experiment, co-inoculation of Streptomyces spp. LuP30 or LuP47B has shown enhance the fixation of atmospheric nitrogen by 47% to 72%, respectively, rather than have any major effect on the uptake of soil N.

The actinobacteria has been also studied using different sources of lucerne rhizobia from two soil extracts and with two more legumes (sub-clover and serradella). The application of either LuP30 or LuP47B to lucerne seeds inoculated with an extract of soil from Urrbrae High School that contained lucerne rhizobia increased significantly the number and mass of nodules and resulted in an increase of shoot dry weight. Shoot dry weight was increased by LuP30 and LuP47B by 16% and 36%, respectively. LuP47B significantly increased the number of nodules and the total nodule mass per plant. Moreover, LuP30 and LuP47B significantly increased the number of nodules and total nodule mass of sub-clover when they were inoculated with Rhizobium WSM 1325. LuP47B also increased the shoot weight and total mass per plant by 25% and 21% respectively 7 weeks after inoculation with the rhizobia.

The 148 well-sporulating cultures were evaluated for their ability to control the growth of fungal root pathogens Rhizoctonia solani AG8 and Pythium irregulare in vitro. Of the 62 cultures that were active against the R. solani, 4 showed strong activity, and 8 were moderately active. Twenty five cultures were active against the P. irregulare, of which 13 were moderately active. In the tube assay, the number of actinobacteria isolates from lucerne which reduced the damage of R. solani AG8 was more than double the number from the three other legumes; 47 isolates (32 from lucerne and 15 from other legumes) showed efficacy in reducing the damage symptoms associated with R. solani AG8. Six isolates (LuP10, LuP30, LuP44, LuP46B, LuP47B and LuP73B) of the 21 tested in pot assay increased by 34% to 47% the total weight of plants that had been inoculated with both rhizobia and the root pathogen R. solani.

All six isolates showing bio-control activity on lucerne were isolated from lucerne roots and have been identified as Streptomyces spp. by 16S rRNA gene sequencing. LuP30 and LuP47B increased the plant growth, nodulation and nitrogen fixation of lucerne plants, and also reduced the root damage caused by R. solani AG8. Based on a full polyphasic taxonomic evaluation in which they were compared in side-by-side comparisons with their two closest type cultures, the conclusion is that both LuP30 and LuP47B are proposed as new species.

Endophytic actinobacteria isolated from roots and nodules of legumes have shown potential for use as microbial inoculants for improving the growth and symbiosis of lucerne. Careful screening and selection of endophytic actinobacteria has provided the most beneficial candidates for co-inoculation with different rhizobial partners and hosts. Field studies are needed to establish if the benefits measured in these studies are confirmed under conditions of more complex micro-flora interactions.

Keywords: Alfalfa; Streptomycete actinomycetes; Nitrogen; Endophytes; Nodulation

Subject: Medical Biotechnology thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Chris Franco