bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 1 Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in 2 the Philippines 3 4 Maria Luisa Tabing Masona,c, Baby Lyn Cortez Tabingd, Akihiro Yamamotob, Yuichi Saekib 5 6 aInterdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 7 Gakuenkibanadai Nishi, Miyazaki, Japan 8 bFaculty of Agriculture, University of Miyazaki, Gakuenkibanadai Nishi, Miyazaki, Japan 9 cCollege of Agriculture, Central Luzon State University, Science City of Muñoz, Nueva 10 Ecija, Philippines 11 dCollege of Agriculture, Don Mariano Marcos Memorial State University-Rosario Campus, 12 Rosario La Union, Philippines 13 14 Running Head: Diversity of bradyrhizobia in the Philippines 15 16 #Address correspondence to Y. Saeki: [email protected]. 17 bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 18 ABSTRACT 19 The diversity of indigenous bradyrhizobia from soils collected at 11 locations in the 20 Philippines was investigated using PSB-SY2 local soybean cultivar as the host plant. 21 Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) 22 treatment for 16S rRNA, 16S-23S rRNA internal transcribed spacer (ITS) region and rpoB 23 housekeeping gene was performed primarily to detect the genetic variation among the 424 24 isolates collected. Then, sequence analysis of 16S rRNA, ITS region and rpoB gene was 25 performed for the representative isolates. Majority of the isolates were classified under 26 Bradyrhizobium elkanii, B. diazoefficiens, B. japonicum, Bradyrhizobium sp., and few 27 isolates were related to B. yuanmingense. Genetic variations observed through PCR-RFLP 28 and sequence analyses of the ITS region and rpoB gene generally occurred in B. elkanii, 29 suggesting an occurrence of gene transfer. Shannon’s diversity index showed varied results 30 with a lowest score of 0.00 and highest at 0.98 indicating a very diverse population of 31 bradyrhizobia across the country. Among all the factors considered in this work, soil 32 management such as period of flooding and some soil properties provided major influence 33 on the distribution and diversity of soybean bradyrhizobia in the country. Thus, it is 34 proposed that the major micro-symbiont of soybean in the Philippines are B. elkanii for bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 35 non-flooded soils, then B. diazoefficiens and B. japonicum for flooded soils. 36 Importance 37 Agriculture production in the Philippines has been and is currently heavily dependent on 38 chemical inputs with mainly rice or corn mono-cropping that it rendered the soil acidic and 39 unproductive. Legume research in the country are mainly focused on plant varietal 40 improvements and very few are aimed at understanding the ecological niche of rhizobia 41 present in the soil. Since soybean has mutual relationship with rhizobia, this legume is a 42 good fallow crop or a rotation crop after rice and corn to help build up the nitrogen stock in 43 the soil. The significance of this research is the better understanding of the ecological niche 44 of indigenous soybean bradyrhizobia, particularly in a tropical archipelago like the 45 Philippines. This work was conceptualized with the utmost goal to increase soybean yield 46 by harnessing and evaluating the indigenous rhizobia in the soil to make production more 47 sustainable and human-friendly. 48 49 KEYWORDS 50 B. elkanii, B. diazoefficiens, B. japonicum, flooded soil, non-flooded, soil management 51 bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 52 INTRODUCTION 53 The genus Bradyrhizobium is a major micro-symbiont of soybean (Glycine max [L.] 54 Merrill) that could form nodule with legumes through symbiosis. The symbiotic 55 relationship between rhizobia and legume is a highly specific, complicated and 56 energy-exhaustive process (50) which may lead to improved crop yield if efficient and 57 sustained. It is considered that Bradyrhizobium is the predominant genus of rhizobia in the 58 tropics (9). Tropical regions have diverse environmental gradients that could influence the 59 diversity of organisms. In this regard, high diversity of soybean-nodulating rhizobia exist in 60 tropical regions (7) than temperate regions. By far, diverse species of bradyrhizobia were 61 reported to be micro-symbionts of soybean such as Bradyrhizobium japonicum, B. elkanii, 62 B. yuanmingense, B. liaoningense, B. huanghuaihaiense and B. diazoefficiens (3, 8, 16, 20, 63 34, 51, 55). Accordingly, many reports stated that soil pH, salinity, climate and cultural 64 management influence the diversity and distribution of soybean bradyrhizobia (1, 5, 11, 22, 65 24, 37, 44, 54). High diversity of microorganisms in the soil plays an important role in 66 maintaining soil health and vast, accurate information about this could lead to better crop 67 productivity. 68 Philippines is a tropical country characterized by high temperature, humidity and bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 5 69 abundant rainfall. It has two pronounced seasons based mainly from rainfall: (1) dry season 70 – from December to May and (2) rainy season – from June to November. Aside from 71 Baguio City, the average temperature in the country is almost similar at 25.5 – 27.5°C. The 72 country’s agricultural production system has been heavily dependent on chemical inputs 73 with rice or corn mono-cropping that it rendered the soil acidic and unproductive. Thus, the 74 need for an effective and efficient symbiosis between legume and rhizobia became more 75 important to increase crop’s yield in a sustainable and environmentally safe production 76 system. Soybean is a good fallow or rotation crop after rice and corn. Although rice is the 77 major agricultural product in the Philippines, recent trend recognized the role of soybean in 78 nutrition and soil fertility restoration. This prompted its Government to create a Research 79 and Development Roadmap for Soybean to increase the area and volume of production. 80 This also paved the way for researchers to improve inoculation techniques to support the 81 government’s endeavor. 82 Yet, inoculation does not always succeed due to several reasons such as incompatibility 83 between the macro and micro-symbiont (12, 52, 53), competition between the indigenous 84 and inoculated rhizobia (15, 42) and the above stated factors that influence the ecology and 85 diversity of rhizobia. Hence, it is necessary to understand the factors influencing the bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 6 86 ecology and diversity of indigenous rhizobia prior to inoculation. 87 Except for one location in the Philippines, which is Nueva Ecija (24), there is no 88 information about the indigenous soybean rhizobia in Philippine soils. Since the Philippines 89 is composed of more than 7,000 islands surrounded by bodies of water with considerable 90 variation in agro-environmental conditions, it is hypothesized that diverse species of 91 bradyrhizobia can be collected and identified to help in improving future inoculation 92 techniques. Therefore, soil samples were collected from 11 locations in the Philippines to 93 obtain information about the diversity and distribution of indigenous soybean bradyrhizobia. 94 A Philippine soybean cultivar was used to trap the indigenous rhizobia from the collected 95 soils then, PCR-RFLP and sequence analysis of the targeted genes were performed. 96 97 MATERIALS AND METHODS 98 Soil Collection 99 Soil samples were collected from eleven locations previously/currently planted with 100 soybean and/or other legumes (Fig. 1). Information on the study sites are summarized in 101 Table 1. Surface litters were removed before obtaining a bar of soil with dimension of 20 102 cm depth and 2 to 3 cm thickness and weighed approximately 1 kg. The 1 kg of composite bioRxiv preprint doi: https://doi.org/10.1101/337394; this version posted June 2, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 7 103 soil sample per location was obtained from mixing and quartering at least 5 sub-samples 104 that weighed approximately 1kg each. Half of the 1 kg composite soil was air dried and 105 pulverized for soil analyses that include pH and electrical conductivity (EC) by 1:2.5 water 106 extraction method, Total C, Total N, Bray P, K (flame spectrophotometer) and soil texture 107 then, the remaining 0.5 kg was freshly used for soybean cultivation. Soil texture was 108 analyzed by Hydrometer method as described previously (4), whereas C and N were 109 analyzed by automatic high sensitive NC Analyzer Sumigraph NC-220F (Sumika Chemical 110 Analysis Service. Ltd., Tokyo, Japan).