Microbial inoculation elicited changes in phyllosphere microbial communities and host immunity suppress Magnaporthe oryzae in a susceptible rice cultivar

Highlights

• Microbes evaluated as foliar spray or soil drench against Magnaporthe oryzae.

• Foliar and soil drench of Bacillus sp. lowered hydrolytic enzymes activities.

• Foliar and soil drench altered nifH and bacterial amoA gene abundances.

• nifH gene copies significantly higher in foliar inoculation of Calothrix sp. (C2).

• Root-shoot continuum important in effective biocontrol using microbes.

Abstract

The hemibiotrophic fungal pathogen Magnaporthe oryzae causes serious crop losses in rice. As phyllospheric microbes share a common habitat with this foliar pathogen, they can be deployed as the change-agents for engineering desired phyllospheric microbial communities to inhibit the disease progression. Comparative evaluation of a set of native microbes, when applied as foliar spray or soil drench was undertaken, showed significant interactive effects on the concentrations of chlorophyll a and b, and carotenoids. A significant enhancement was recorded in the activities of chitosanase, β-1,3-endoglucanase and β-1,4-endoglucanase (CMCase) in the disease challenged plants, as compared to healthy plants. Among the treatments, Bacillus sp. (B1) inoculation recorded lowest values of the activities of all three hydrolytic enzymes, while Bacillus sp. (B4) and Nostoc-Anabaena consortium (C1) led to 30–60% decreases, both as foliar and soil drench modes of application. Distinct changes in the abundances of eubacterial and phylum Cyanobacterial 16S rRNA gene copies, nifH and bacterial amoA illustrated the significance of foliar over the soil drench method. The nifH gene copies were significantly higher due to the foliar method of Calothrix sp. (C2) inoculation, and values were significantly at par values with both the soil drench application of B1 (Bacillus sp.) and C1 (Nostoc-Anabaena consortium) treatments. This study illustrates the significance of the root-shoot linkages in the effective biocontrol of these promising, indigenous microbes, applied as soil drench or foliar agents, which can be useful in abating the incidence of the fungal pathogen in an environment –friendly manner.

Introduction

In nature, plants are known to shape their microbiomes, which in turn, influence their biochemical and physiological activities [1]. The phyllosphere representing the aerial parts of the plant comprises organisms of various taxonomic kingdoms such as archaea, eubacteria, filamentous fungi, protozoa, cyanobacteria, and even nematodes with diverse functionalities [2]. However, this niche has been investigated mainly in terms of its significance in carbon assimilation, besides scattered reports on nitrogen fixation [3]. This is mainly, as the phyllosphere has to bear brunt of extremes of pest attacks, or weather and other abiotic factors, including radiations, leading to colonization by only a select group of robust organisms [4]. In our earlier studies, cultivation methods and fertilizer application were found to modulate the distribution of microbial taxa and functional attributes of the phyllosphere microbiome in a popular rice variety grown in tropical sandy loam soil [5,6].

Cultivated rice (Oryza sativa L.) is one of the most important food crops nourishing half of the human population globally, and accounting for 23% of the world's calorie intake [7]. Among fungal diseases causing severe losses in rice, Magnaporthe oryzae causing rice blast ranks among the most devastating [8]. Being a foliar disease, it may have significant implications on the phyllosphere microbiome, and bring about more than 50% losses. The genome sequence of M. oryzae is now available [9] and hundreds of genes involved in its pathogenesis have been identified using functional genomic approaches. The management of rice blast disease has generally involved the cultural and chemical methods, besides the breeding approaches. The use of fungicides which is extensive in many rice growing regions for the management of blast disease is not only polluting these environments, but also leads to development of resistance in pathogens along with impacting indigenous communities [10].

Several plant growth promoting microorganisms (PGPMs) with biocontrol efficacy have been isolated from rhizosphere or soil and used against blast disease [11,12]. Most of these microorganisms modulate the plant's physiology, eliciting changes in the defence, pathogenesis and antioxidant enzyme machinery [13], while plant and soil type are known to influence the structure and functioning of rhizosphere microbiome [14,15]. Recent reports suggest the existence of linkages between below-and above ground parts of the plant, which includes a diverse array of bacteria, fungi, some of which are transient members, while some may become endophytes [16]. In order to investigate this aspect in terms of disease suppressive potential, inoculation of bacteria/cyanobacteria having plant growth promoting and biocontrol potential was done, using both foliar and soil drench modes of application independently. The present study focuses on evaluating the phyllosphere isolates as potential biocontrol agents against blast disease caused by M. oryzae and identifying the most promising option for use in disease management strategies with already recommended practices. Analyses of the enzyme machinery elicited, leaf pigments and nutrients, and quantification of the leaf microbial abundance and diversity, as well as functional genes - nifH and bacterial amoA were undertaken. The genes selected-nifH encodes for the dinitrogenase reductase which is a subunit of the enzyme nitrogenase whereas amoA gene encodes for α subunit of ammonia monooxygenase which catalyses ammonia oxidation. These genes were targeted as nitrogen is limiting in the phyllosphere, while nitrogen is abundant in the atmosphere, and ammonia oxidation is a less investigated phenomenon in the phyllosphere.

Our hypothesis was that inoculation with native phyllospheric microbes, possessing biocontrol ability, may prove promising options in disease suppression, through their beneficial interactions with the host, including those related to nutrient (N) dynamics.