Improving Cowpea production in North West Province using endophytic bacteria and fungi

Abstract

Providing adequate, nutritious and safe food for human race is a huge challenge in the light of the rising cost of living, corona virus pandemic, climatic changes, high energy cost and war. The means of taken care of the global food supply is through the application of agrochemicals that are costly, non-renewable and destructive to the ecological balance. However, this highly intensive agrochemicals production system is not agro-ecologically sustainable. Therefore, there is ongoing efforts by various stakeholders in the agricultural sector to search for substitutes/alternatives to synthetic agrochemicals to achieve sustainable agro-ecological production system. A veritable means of achieving sustainable agroecological production is through the use of microbial resources that are renewable and eco-friendly. Endophytic microbiota research is on the upward trend as important partner in the plant-microbiome production system. Endophytic bacterial and fungal inoculants have shown good potentials in improving plant growth as biofertilizing, bio-stimulating, and bio-pesticidal renewable microbial resources with prospects in attaining agro-ecological balanced and sustainable crop improvement. Cowpea known scientifically as Vigna unguiculata L. Walps is multi-beneficial, highly valuable leguminous crop. It is a crop with high value-addition to human nutritional, animal husbandry sector and also agro-ecological benefits/soil health improvement. However, it is still an under-developed/neglected leguminous crop despite its numerous benefits. Although the African continent remains the major producer of this leguminous crop, a lot of challenges still persists in limiting cowpea optimum productivity and includes abiotic and biotic constraints, such as pest infestations, climatic weather variations among others. A veritable means of changing this narrative of low productivity of cowpea in Africa is by adopting sustainable agro-ecological microbial biotechnology approaches. Hence, the aim of this study is to assess the prospect of using endophytic bacteria and fungi to improve cowpea production yield in North West Province: a semi-arid zone of South Africa. Methodology: Healthy looking and disease-free cowpea plant aerial parts and root zone tissues were collected from farms in North west province, South Africa. Also, cowpea seeds cultivars PAN311 and Bechuana white were collected from Agricultural Research Council, South Africa for the purpose of this study. Standard approaches were deployed in the storage, processing, isolation and authentication of endophytic bacteria and fungi from the cowpea seeds and tissues through morphological and molecular techniques.

The morphometrics methodology that was deployed in characterizing the endophytic microbiota include Gram staining techniques, sugar utilization, varied biochemical test and growth at different temperature for the endophytic bacteria. For the fungi isolates, macroscopic growth characteristics on different mycological growth media as well as microscopic features were deployed for morphological identification. For the molecular authentication, genomic extraction kits were used for extracting the genomic DNA, polymerase chain reaction (PCR) amplification protocol using both bacteria and fungi primers, was used to amplify 16S rRNA gene for the bacteria isolates, and ITS rRNA gene was used to amplify the fungi isolates. Agarose gel electrophoresis techniques was used in checking the quality of amplicons for both bacteria and fungi isolated. The amplicons were sequenced in both the forward and reverse directions. Different bioinformatics software were used to trim, clean and edit the sequence chromatograms before comparing it with existing sequences through BLAST search of the national center for biotechnology information (NCBI) database platform. Phylogenetic tree was then constructed using MEGA 7 software. Thereafter, plant growth promoting assay procedures were deployed in assessing the plant growth-improving attributes of both bacteria and fungi isolates. The various plant growth promoting attributes assay determined include: exopolysaccharide production, ammonia production, siderophore production, biofilm forming potential, hydrogen cyanide producing traits, solubilization of phosphate traits, as well as 1-aminocyclopropane-1-carboxylic acid deaminase capability. In addition to their invitro growth promoting potential experiment, growth chamber studies were carried out to assess the in-planta growth promoting capability of the endophytic isolates. To further elucidate the mechanistic approaches used by the highly effective endophytic microbiota isolated, selected isolates that included 2 bacterial isolates and a fungal isolate were subjected to in-depth genomic exploration using next generation whole genome sequencing techniques. More so, field experimental planting of two cultivars of cowpea was done over 2 planting seasons in Mafikeng, South Africa. A 2 X 5 factorial experimental design were randomized in completely block layout with three replications. The treatments deployed through seed biopriming with endophytic bacteria and fungi culture were applied as a single bioinoculant and as mixed bioinoculants. The control treatments were cowpea cultivars planted without inoculation with endophytic microbiota. Agronomic yield and growth parameters data were monitored and subjected to statistical analysis using SPSS version 20 was deployed for analysis of data. Results: The results indicated that 33 endophytic bacteria isolates were morpho-genetically authenticated, while 26 fungi isolates were also characterized. The 33

endophytic bacteria strains are diverse in terms of taxonomy, molecular and metabolic characteristics and falls into 11 genera that include: Bacillus, Lynsinibacillus, Pseudomonas, Staphylococcus, Paenibacillus, Mammalicoccus, Brevundimonas, Stenotrophomonas, Myroides, Micrococcus and Ignatzscineria. All the endophytic bacteria isolates possess multifarious invitro plant growth improving attributes and improves cowpea seeds and seedling germination. Likewise, the 26 fungal isolated strains possess multiple plant-growth improving attributes. Three endophytic microbiota showing high-level of bioactivity were selected for further genomic exploration and then deployed in cowpea field experimentation. They are Stenotrophomonas geniculata NWUBe21 and Pseudomonas carnis NWUBe30, as well as Aspergillus minisclerotigenes NWUFe10. The whole genome exploration studies indicated that these cowpea endophytic isolates possess plant growth improving genes and also vital secondary metabolites gene clusters. The field studies data showed that both single and mixed culture endophytic inoculants enhanced cowpea agronomic yield and growth parameters significantly compared to the un-inoculated control. Conclusion: The results of this findings have shown that endophytic microbiota from cowpea plants are potential renewable microbial resources that could be deployed in cowpea production for productivity enhancement in order to achieve sustainable agroecological production of this vital leguminous crop.