New method for arbuscular mycorrhizal fungus spore separation using a microfluidic device based on manual temporary flow diversion.

Arbuscular mycorrhizal fungi are beneficial components often included in biofertilizers. Studies of the biology and utilization of these fungi are key to their successful use in the biofertilizer industry. The acquisition of isolated spores is a required step in these studies; however, spore quality control and spore separation are bottlenecks. Filtered and centrifuged spores have to be hand-picked under a microscope. The conventional procedure is skill-demanding, labor-intensive, and time-consuming. Here, we developed a microfluidic device to aid manual separation of spores from a filtered and centrifuged suspension. The device is a single spore streamer equipped with a manual temporary flow diversion (MTFD) mechanism to select single spores. Users can press a switch to generate MTFD when the spore arrives at the selection site. The targeted spore flows in a stream to the collection chamber via temporary cross flow. Using the device, spore purity, the percentage of spore numbers against the total number of particles counted in the collecting chamber reached 96.62% (median, n = 10) which is greater than the spore purity obtained from the conventional method (88.89% (median, n = 10)).

Empowering rice seedling growth by endophytic Bradyrhizobium sp. SUTN9-2.

Bradyrhizobium sp. strain SUTN9-2 was confirmed as rice endophytic bacteria and also as rice growth promotion agent. SUTN9-2 showed the capability of plant growth promotion characteristics, such as indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase productions and nitrogen fixation. In this study, the ability of SUTN9-2 to stimulate rice growth was investigated at different stages with N-free and NH4 NO3 under in vivo condition. The rice dry weight and chlorophyll content could be enhanced when SUTN9-2 was inoculated in N-free, especially at seedling stage (7 and 14 dai). The rice dry weight was also increased when SUTN9-2 was inoculated with NH4 NO3 at 7 and14 dai. The results of quantitative analysis of IAA and ACC deaminase were inconsistent with the expression of genes involved in IAA (nit) and ACC deaminase (acdS) productions. This inconsistently could implied that IAA and ACC deaminase produced from SUTN9-2 do not directly affect rice growth, but other factors resulting from the production of IAA and ACC deaminase could be involved. Moreover, the expression of genes involved in nitrogen fixation (nifH and nifV) of SUTN9-2 was also induced in rice tissues. This finding suggested that rice growth promotion may be supported by NH4 NO3 together with nitrogen fixation by SUTN9-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Indole-3-acetic acid, 1-amino-cyclopropane-1-carboxylic acid deaminase productions and nitrogen fixation may play important roles in rice growth promotion by endophytic SUTN9-2, especially at early rice seedling growth stage, which has the potential to be used as rice seedling growth promoter in the system of rice intensification.

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP).

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol-chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.

Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume.

Bradyrhizobium is an endophytic bacterium under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system. Monitoring and bio-imaging of this nitrogen fixing bacterium is essential for the study of plant-microbe evolution, soil microbiome, as well as quality control in organic farming. While phage display antibody technology has been widely used to generate recombinant antibody for myriad medical purposes, so far, this technology has been minimally applied in the agricultural sector. In this study, single-chain variable fragments (scFv) against two Bradyrhizobium strains SUTN9-2 (yiN92-1e10) and DOA9 (yiDOA9-162) were isolated from a human phage display antibody library. Specific binding of scFv was demonstrated by ELISA and confocal-immunofluorescence imaging techniques. Bradyrhizobium localization in both endophytic and bacteroid forms could be observed inside rice tissue and plant nodule, respectively. Moreover, successful application of the recombinant antibody for the evaluation of nodule occupancy was also demonstrated in comparison with standard GUS-staining method. The results of this study showed for the first time the potential use of human phage display scFv antibody for imaging and monitoring of Bradyrhizobium biofertilizer and thus could be further applied for point-of-detection of bacterial inoculum in the legume-rice rotational crop system. IMPORTANCE Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively. These two recombinant scFv antibodies could be used for precise detection of the rhizobia both in symbiosis with legume and endophyte in rice tissue by ELISA and immunofluorescent staining, during legume-rice rotational cropping system in the field. This methodology can be further employed for the study of other plant-microbe interactions and monitoring of biofertilizer in diverse sustainable cropping systems as well as in precision agriculture.