Endophytic bacteria associated with bananas(Musa spp.) in Kenya and their potential as biological fertilizers

Abstract

Bananas and plantains are of special significance to human societies being the fourth most important food in the world today after rice, wheat, and maize. In Kenya, banana is the most popular fruit. In central and western Kenya, the cooking banana forms part of the staple food. Increased trade in local, regional and international markets has also made banana an important cash crop, and in some cases the only source of income for rural populations. Banana production in Kenya is however much below its potential with an estimated average yield of 19 tonnes per hectare as opposed to an average potential yield of 35-45 tonnes per hectare. Several factors including declining soil fertility contribute to this low productivity. The most obvious solution to declining soil fertility is application of mineral fertilizers. However, these are expensive and out of reach for most resource-poor farmers, who constitute the vast majority of banana farmers in Kenya. Additionally, use of chemical fertilizers is not environment friendly. An alternative approach is to improve nutrient uptake by plants through utilization of microbial inoculants. Endophytes, as microbial inoculants, are increasingly gaining scientific and commercial interest because of their potential to improve plant quality and growth and their close association with internal tissues of host plant. To the author’s knowledge, endophytic bacteria of bananas in Kenya have not been isolated or identified and consequently there is limited information on their diversity and their functional potential in regard to banana growth and nutrition. Four main studies were undertaken. In the first study bacteria were isolated from roots, corms and stems of two banana cultivars (Musa AAA – Cavendish &Musa AAB – plantain) collected from five different geographical regions (Juja, Maragua, Embu, Meru and Kisii).With five different isolation media, a total of 2,717 isolates were obtained.Morphological characterization was done on the basis of colony color, appearance, motility and Gram staining; and based on the colony morphotypes 214 representative isolates were selected.Biochemical tests done included gelatinase test, starch hydrolysis, catalase production, nitrate reduction and citrate and sugar utilization. The isolates were profiled using the whole-cell matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF/MS). Proteins from the living cells wereextracted using the ethanol/formic acid extraction procedure, and intact molecular weights of the ionized proteins and the pattern of the protein molecular weights were used as fingerprints.Forty three isolates were selected for partial 16S rRNA gene sequencing, whichgrouped them into three families Enterobacteriaceae, Pseudomonadaceae and Bacillaceae. The family Enterobacteriaceae was the most diverse with 8 genera namely Serratia (17 isolates), Rahnella (4 isolates), Enterobacter (2 isolates), Yokenella (2 isolates), Raoultella (2 isolates), Klebsiella (1 isolate), Yersinia (1 isolate) and Ewingella (1 isolate). Both the Pseudomonadaceae and Bacillaceae families were represented by only one genus, that is,Pseudomonas (12 isolates) and Bacillus (1 isolate), respectively.Serratia and Pseudomonas species were the most abundant with 17 isolates and 12 isolates, respectively. Of the 10 genera identified, Rahnella, Yokenella, Raoultella, Yersinia andEwingellahad not been previously described as endophytic in banana plants. Full-length sequencing allowed identification of some five selected isolates as Bacillus subtilis subsp. inaquosorum, Enterobacter ludwigii, Ewingella americana, Rahnella aquatilis and Flavimonas oryzihabitans and the sequence data generated have been deposited with the NCBI GenBank under accession numbers AB675632 to AB675636.Of significance is that isolate K22V1c is being proposed as novel having showed a similarity value of less than 97% (95.27%) with its closest relative Klebsiella granulomatis. The second study characterized the 43 isolates on the basis of their in-vitro plant growth-promoting activities that included ability to fix free nitrogen, solubilize phosphates and produce siderophores. All the 12 Pseudomonas isolates showed potential for siderophore production with Flavimonas oryzihabitansisolates showing the highest potentialas determined on blue Chrome Azurol S (CAS) agar plates. Twenty seven isolates were observed to solubilize phosphates, with Rahnella isolates showing the highest potentialas determined on NBRIP growth medium. All the isolates grew on solid nitrogen-source free medium and had varied levels of acetylene reduction activity, suggesting their ability to fix atmospheric nitrogen.PCR amplification of the nifH gene, which codes for the enzyme nitrogenase reductase, was attempted without much success. In the third study, tissue-cultured banana plantlets (Musa spp. cv. Grande Naine) were inoculated with some selected isolates and effects of inoculation on plant growth, physiology and mineral nutritionat different fertilizer regimes were investigated. Ewingella americana(K32V2c)showed a positive significant effect (P=0.05)on the relative change in plant height and number of green leaves whileEnterobacter ludwigii(J1V1r)significantly (P=0.05) increased shoot dry weight. Inoculation of plants with Bacillus subtilis subsp. Inaquosorum(M9V1r), Enterobacter ludwigii(J1V1r)and Ewingella americana(K32V2c)significantly (P=0.05) increased chlorophyll content in plants while Rahnella aquatilis(ME19V2c) increased P concentration in plant shoots though not significantly. The fourth study sought to confirm the endophytic habitat and colonization ability of the isolated bacteria. Endophytic habitat of Flavimonas oryzihabitans(K50V2s) was confirmed through microscopic evidence of “tagged” bacteria inside plant tissues.The green fluorescent protein reporter gene (mTn5gusA-pgfp21) from E. coli S17-1was used to tag the bacteria while fluorescence scanning was done under theConfocal Laser Scanning Microscope.Onlyconjugation of Flavimonas oryzihabitans (K50V2s) with E. coli S17-1 (mTn5gusA-pgfp21) resulted to successful transconjugants. In conclusion, endophytic bacteria of bananas in Kenya were successfully isolated and identified as Serratia, Pseudomonas, Rahnella, Enterobacter, Yokenella, Raoultella, Klebsiella, Yersinia, Ewingella and Bacillus species.Rahnella, Yokenella, Raoultella, Yersinia andEwingellaspecies having not been reported in association with bananas by the time of this study suggests a large richness of banana endophytic bacterial species than has so far been reported.It’s now clear that many diazotrophic microbes inhabit the tissues of banana plants and there is the potential of exploiting them once conditions for their use is optimized. Rahnella aquatilis (ME19V2c and ME18V2c) and Flavimonas oryzihabitans (K50V2s)having showedability tosolubilize phosphate and produce siderophore, respectively and also ability to fix free nitrogen could be proposed as potential biofertilizers for sustainable banana production in Kenya.Bacillus subtilis subsp. inaquosorum (M9V1r), Enterobacter ludwigii (J1V1r), Ewingella americana(K32V2c), Rahnella aquatilis (ME19V2c) and Flavimonas oryzihabitans (K50V2s)have potential to promote plant growth. However, to facilitate their use in practical agronomic production, reliable and practical methods of inoculum delivery must be developed.