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Abstract An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases. Key points • Strain A9 can secrete a thermostable β-glucosidase that has high glucose tolerance • A coculture of strain A9 and C. thermocellum showed high cellulose degradation • Strain A9 achieves biological saccharification without addition of β-glucosidase

D-Psicose is a rare, low-calorie sugar that is found in limited quantities in national products. Recently, D-psicose has gained considerable attention due to its potential applications in the food, nutraceutical, and pharmaceutical industries. In this study, a novel D-psicose 3-epimerase (a group of ketose 3-epimerase) from an extremely halophilic, anaerobic bacterium, Iocasia fonsfrigidae strain SP3-1 (IfDPEase), was cloned, expressed in Escherichia coli, and characterized. Unlike other ketose 3-epimerase members, IfDPEase shows reversible epimerization only for D-fructose and D-psicose at the C-3 position but not for D-tagatose, most likely because the Gly218 and Cys6 at the substrate-binding subsites of IfDPEase, which are involved in interactions at the O-1 and O-6 positions of D-fructose, respectively, differ from those of other 3-epimerases. Under optimum conditions (5 µM IfDPEase, 1 mM Mn2+, 50 °C, and pH 7.5), 36.1% of D-psicose was obtained from 10 mg/mL D-fructose. The IfDPEase is highly active against D-fructose under NaCl concentrations of up to 500 mM, possibly due to the excessive negative charges of acidic amino acid residues (aspartic and glutamic acids), which are localized on the surface of the halophilic enzyme. These negative charges may protect the enzyme from Na+ ions from the environment and result in the lowest pI value compared to those of other 3-epimerase members. Moreover, without adjusting any ingredients, IfDPEase could improve coconut water quality by converting D-fructose into D-psicose with a yield of 26.8%. Therefore, IfDPEase is an attractive alternative to enhancing the quality of fructose-containing foods.

We isolated and analysed a Gram-negative, facultatively thermophilic, xylan-degrading bacterium that we designated as strain DA-C8T. The strain was isolated from compost from Ishigaki Island, Japan, by enrichment culturing using beech wood xylan as the sole carbon source. The strain showed high xylan degradation ability under anaerobic growth conditions. The isolate grew at 37–60 °C (optimum, 55 °C) and pH 4.0–11.0 (optimum, pH 9.0). As well as xylan, strain DA-C8T could use polysaccharides such as arabinoxylan and galactan as carbon sources. Comparison of 16S rRNA gene sequences indicated that strain DA-C8T was most closely related to Paenibacillus cisolokensis LC2-13AT (93.9 %) and Paenibacillus chitinolyticus HSCC596 (93.5 %). In phylogenetic analysis, strain DA-C8T belonged to the same lineage as Xylanibacillus composti K13T (92.5 %), but there was less statistical support for branching (70 %). Digital DNA–DNA hybridization, average nucleotide identity values and average amino acid sequence identity between strain DA-C8T and P. cisolokensis LC2-13AT were 21.8, 68.3 and 58.2 %, respectively. Those between strain DA-C8T and X. composti K13 were 23.7, 67.7 and 57.6 %, respectively. The whole-genome DNA G+C content of strain DA-C8T was 52.3 mol%. The major cellular fatty acids were C16 : 0 (42.9 %), anteiso-C15 : 0 (20.0 %) and anteiso-C17 : 0 (16.7 %), the major quinone was menaquinone 7, and the major polar lipids were unidentified glycolipids. On the basis of phenotypic, chemotaxonomic and phylogenetic evidence, a novel genus is proposed—Insulambacter gen. nov.—for the novel species Insulambacter thermoxylanivorax sp. nov. The type strain is DA-C8T (=JCM 34211T=DSM 111723T).

Key message Status of the current outbreak of cassava mosaic disease (CMD) in Southeast Asia was reviewed. Healthy cassava seed production and dissemination systems have been established in Vietnam and Cambodia, along with integrated disease and pest management systems, to combat the outbreak. Abstract Cassava (Manihot esculenta Crantz) is one of the most important edible crops in tropical and subtropical regions. Recently, invasive insect pests and diseases have resulted in serious losses to cassava in Southeast Asia. In this review we discuss the current outbreak of cassava mosaic disease (CMD) caused by the Sri Lankan cassava mosaic virus (SLCMV) in Southeast Asia, and summarize similarities between SLCMV and other cassava mosaic begomoviruses. A SATREPS (Science and Technology Research Partnership for Sustainable Development) project “Development and dissemination of sustainable production systems based on invasive pest management of cassava in Vietnam, Cambodia and Thailand”, was launched in 2016, which has been funded by The Japan International Cooperation Agency (JICA) and The Japan Science and Technology Agency (JST), Japan. The objectives of SATREPS were to establish healthy seed production and dissemination systems for cassava in south Vietnam and Cambodia, and to develop management systems for plant diseases and insect pests of cassava. To achieve these goals, model systems of healthy seed production in Vietnam and Cambodia have been developed incorporating CMD-resistant planting materials through international networks with The International Center for Tropical Agriculture (CIAT) and The International Institute of Tropical Agriculture (IITA).

Oil palm empty fruit bunch (OPEFB) is lignocellulosic waste from the palm oil industry in Southeast Asia. It is difficult to degrade because of its complex matrix and recalcitrant structure. To decompose OPEFB, highly efficient micro-organisms and robust enzymatic systems are required. A bacterium with high degradation ability against untreated OPEFB was isolated from earthworm soil biofertilizer and designated as strain EW123T. Cells were Gram-stain-positive, rod-shaped and catalase-positive. In tests, the strain was negative for mycelium formation, motility, nitrate reductase and urease. The 16S rRNA gene analysis of the isolate showed 98.21 % similarity to Cellulomonas uda NBRC 3747T, whereas similarity to other species was below 98 %. The genome of strain EW123T was 3 834 009 bp long, with 73.97 mol% G+C content. Polar lipid analysis of strain EW123T indicated phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol and aminophospholipid as the lipid components of the cell wall. The major cellular fatty acid was anteiso-C15 : 0 (41.26 %) and the isomer of 2,6-diaminopimelic acid (DAP) was meso-DAP. The average nucleotide identity value between the genome sequences of EW123T and C. uda NBRC 3747T was 88.6 %. In addition, the digital DNA–DNA hybridization and genome average amino acid between those strains were 36.1 and 89.68 %, respectively. The ORF number (186) of carbohydrate-active enzymes, including cellulases, xylanases, mannanase, lipase and lignin-degrading enzymes, was higher than those of related strains. These results indicate that the polyphasic characteristics of EW123T differ from those of other related species in the genus Cellulomonas . We therefore propose a novel species of the genus Cellulomonas , namely Cellulomonas palmilyticum sp. nov. (type strain TBRC 11805T=NBRC 114552T), with the ability to effectively degrade untreated OPEFB.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

A tobamovirus was isolated from leaves of Scopolia japonica that had mild yellowing. Back-inoculation of healthy Scopolia japonica with the isolated virus induced mild mottle on upper leaves. Phylogenetic analysis of the coat protein and replicase protein revealed that the newly isolated tobamovirus is related to yellow tailflower mild mottle virus. The highest identity of the 6350-nucleotide sequence of the whole genome shared with that of other tobamoviruses was 71%. According to species demarcation criterion for the genus Tobamovirus, our result suggests that scopolia mild mottle tobamovirus is a new species within the genus Tobamovirus.

A tobamovirus was isolated from leaves of a Scopolia japonica plant showing mild yellowing. Back-inoculation of healthy Scopolia japonica with the isolated virus induced mild mottle on upper leaves. Phylogenetic analysis based on coat protein and replicase protein sequences revealed that the newly isolated tobamovirus was most closely related to yellow tailflower mild mottle virus (YTMMV). The newly isolated tobamovirus shared the highest nucleotide sequence identity (71%) with YTMMV, which is lower than the cutoff (90%) set for species demarcation in the genus Tobamovirus. Thus, our result suggested that scopolia mild mottle virus (SMMoV) is a new tobamovirus that infects Scopolia japonica plants in Japan.

To discover more efficient degradation processes of lignocellulosic biomass, it is still important to analyze genomic and enzymatic data from bacteria that have strong xylanolytic ability. Here, we present the draft genome sequences of the xylanolytic bacteria Paenibacillus cisolokensis strain LC2-13A and Xylanibacillus composti strain K-13 that are closest to Paenibacillus sp. strain DA-C8, which has strong xylan degradation ability under anaerobic growth conditions. Whole-genome sequencing on the Ion GeneStudio S5 System yielded 277 contigs with total size 5,305,208 bp and G+C content 52.3 mol% for strain LC2-13A and 115 contigs with total size 4,652,266 bp and G+C content of 56.2 mol% for strain K-13. The LC2-13A genome had 5,744 protein-coding sequences (CDSs), 57 tRNAs, and 4 clustered regularly interspaced short palindromic repeats (CRISPRs), and the K-13 genome had 4,388 CDSs, 1 rRNA gene, 45 tRNAs, and 5 CRISPRs. The CDSs of LC2-13A and K-13 encoded the following carbohydrate-active enzymes: 98 and 67 glycoside hydrolases, 31 and 29 glycosyl transferases, 23 and 17 carbohydrate esterases, and 13 and 37 carbohydrate-binding modules, respectively. The whole-genome sequences of LC2-13A and K-13 have been deposited in DDBJ/ENA/GenBank under accession numbers BOVK00000000 and BOVJ00000000. The versions described in this paper are version 1.

Cassava mosaic disease (CMD), a serious threat to cassava production, is caused by two begomovirus species in Asia, Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). Cassava mosaic disease has recently spread into Southeast Asia and it is urgent to establish an efficient system to monitor its spread. Cassava fields were surveyed for CMD on the web using a smartphone-based farming support application, AGRIBUDDY, to obtain photos of CMD and their time, location, and contact information posted to the AGRIBUDDY cloud by farmers. To diagnose a pathogen, we designed novel PCR primers for CMD identification. By combining these two techniques, we detected SLCMV occurrence in Kampong Thom, Cambodia.

The screening, identification, and study of the functional properties of cellulolytic xylanolytic bacteria are crucial for the construction of applicable bioprocesses. The thermophilic facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211=DSM111723) exhibiting efficient xylan degradation was newly isolated from compost. Strain DA-C8 completely degraded 1% beechwood xylan within 4 days under anaerobic conditions. By 16S rRNA gene sequence homology and phylogenetic tree analysis, strain DA-C8 was closely related to Paenibacillus cisolokensis and Xylanibacillus composti; however, the average nucleotide identity and digital DNA-DNA hybridization values based on genome information and the carbon source utilization properties indicated that strain DA-C8 belongs to Paenibacillus rather than Xylanibacillus. The gene numbers of xylanase and endoglucanase of strain DA-C8 and X. composti were not different; however, strain DA-C8 had higher abundance of α- L -arabinofuranosidase, β-xylosidase, and β-glucosidase than X. composti. Strain DA-C8 showed decreased xylan and corn hull degradation abilities and growth on xylan medium under aerobic conditions. Quantitative PCR showed high expression of xylan and cellulose degradation genes under anaerobic conditions, but the genes were repressed under aerobic conditions, indicating that strain DA-C8 controls polysaccharide degradation depending on the aeration conditions. Strain DA-C8 is a new species of Paenibacillus with a unique polysaccharide degradation system.

A new badnavirus, aucuba ringspot virus (AuRV), was identified in plants of Aucuba japonica showing mild mosaic, vein banding, and yellow ringspot symptoms on the leaves. The complete nucleotide sequence of the AuRV genome was determined and found to be 9,092 nt in length, and the virus was found to have a genome organization typical of members of the genus Badnavirus. ORF3 was predicted to encode a polyprotein containing conserved movement protein, coat protein, aspartic protease, reverse transcriptase (RT), and RNase H domains. Phylogenetic analysis suggested that this virus is most closely related to codonopsis vein clearing virus but belongs to a distinct species, based on only 69.6% nucleotide sequence identity within the part of ORF 3 encoding the RT and RNase H domains. The vector of AuRV is unknown, but based on phylogenetic relationships, it is predicted to be a type of aphid.

Chitin is the second most abundant organic compound in nature. Although mesophilic bacteria degrade insoluble chitin, there is a paucity of data describing degradation of insoluble chitin by anaerobic thermophilic bacteria. In this report, we screened cow manure compost for new chitin degradation systems, and identified a chitinolytic bacterial community (CBC) that showed high chitin degradation activity under thermophilic conditions, i.e., 1% (w/v) chitin powder degraded completely within 7 days at 60 °C. Metagenomic analysis revealed that the CBC was dominated by two bacterial genera from Hydrogenispora, an uncultured taxonomic group, and Tepidanaerobacter. Hydrogenispora were abundant in the early-to-mid stages of culturing with chitin, whereas the population of Tepidanaerobacter increased during the later stages of culturing. Strains UUS1-1 and GT38, which were isolated as pure cultures using the roll-tube method with colloidal chitin, N-acetyl- d -glucosamine, and glucose as carbon sources, were found to be closely related to H. ethanolica and T. acetatoxydans, respectively. Strain UUS1−1 readily degraded chitin and is the first anaerobic thermophilic chitinolytic bacterium reported, whereas strain GT38 showed no chitinolytic activity. Based on phylogenetic analysis, UUS1−1 and GT38 should be classified as novel genera and species. Zymogram analysis revealed that UUS1−1 produces at least two chitinases with molecular weights of 150 and 40 kDa. A coculture of UUS1−1 and GT38 degraded crystalline chitin faster with lower accumulation of lactate compared with UUS1−1 alone, indicating that the strains maintained a symbiotic association through assimilation of organic acids in chitin degradation and that strain GT38 consumed end-products to reduce end-product inhibition and enhance the degradation of crystalline chitin.

Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60-65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.

Oil palm trunks (OPT) are logged for replantation and the fiber residues are disposed of into the palm plantation area. The fiber residues are expected to increase soil fertility through recycling of carbon and minerals via fiber decomposition. This study investigated the effects of OPT fiber disposal and other lignocellulosic biomass on plant growth and microbial diversity in the soil environment. Four treatment plots were tested: (A) soil+OPT fiber (1:20), (B) soil+sugarcane bagasse (1:20), (C) soil+cellulose powder (1:20), and (D) unamended soil as a negative control. Low plant height, decreased chlorophyll content, and low biomass was observed in corn grown on soil mixed with OPT fiber, cellulose, and sugarcane bagasse, when compared with those of the control. The plants grown with OPT fiber were deficient in total nitrogen and magnesium when compared with those without fiber amendment, which suggested that nitrogen and minerals in soil might be taken up by changing microflora because of the OPT fibers presence. To confirm differences in the soil microflora, metagenomics analysis was performed on untreated soil and soil from each lignocellulose treatment. The microflora of soils mixed with OPT fiber, cellulose and sugarcane bagasse revealed substantial increases in bacteria such as families Cytophagaceae and Oscillospiraceae, and two major fungal genera, Trichoderma and Trichocladium, that are involved in lignocellulose degradation. OPT fiber resulted in a drastic increase in the ratios and amounts of Trichocladium in the soil when compared with those of cellulose and sugarcane bagasse. These results indicate that unregulated disposal of OPT fiber into plantation areas could result in nutrient loss from soil by increasing the abundance of microorganisms involved in lignocellulose decomposition.

A novel Gram-negative, spore forming, obligately anaerobic, thermophilic, chitin-degrading bacterium, designated UUS1-1T, was isolated from compost on Ishigaki Island, Japan by enrichment culturing using chitin powder as the carbon source. The strain has unique, long, hair-like rod morphological features and exhibits strong degradation activity toward crystalline chitin under thermophilic conditions. Growth of the novel strain was observed at 45–65 °C (optimum, 55 °C) and pH 6.5–7.5 (optimum, pH 7.0). In addition to chitin, the strain utilized several other carbon sources, including N-acetylglucosamine, glucose, galactose, mannose, maltose, cellobiose, fructose and sucrose. The end products of chitin degradation were acetate, lactate, H2 and CO2. Phylogenetic tree analysis based on 16S rRNA gene sequences revealed a clear affiliation of the proposed bacterium to the phylum Firmicutes ; the most closely related species were Hydrogenispora ethanolica LX-BT and Desulfotomaculum thermobenzoicum DSM6193T with similarities of 90.4 and 87.8 %, respectively. The G+C content of the genomic DNA was 52.1 mol%. The average nucleotide identity and digital DNA–DNA hybridization values between the genomes of UUS1-1T and H. ethanolica LX-BT were 65.5 and 21.0 %, respectively. The cellular fatty acid composition of the strain was C16 : 0, anteiso-C15 : 0, C14 : 0, C12 : 0 3-OH and dimethyl acetal-C13 : 0. Based on phenotypic, chemotaxonomic and genotypic analysis, strain UUS1-1T represents a novel genus and species, for which the name Capillibacterium thermochitinicola gen. nov., sp. nov. is proposed. The type strain is UUS1-1T (=JCM 33882T=DSM 111537T).

Thermophilic, facultatively anaerobic, xylanolytic bacterial strain DA-C8 (=JCM34211 =DSM111723), newly isolated from compost, shows strong beechwood xylan degradation ability. Whole-genome sequencing of strain DA-C8 on the Ion GeneStudio S5 system yielded 69 contigs with a total size of 3,110,565 bp, 2,877 protein-coding sequences, and a G+C content of 52.3 mol%. Genome annotation revealed that strain DA-C8 possesses debranching enzymes, such as β-L-arabinofuranosidase and polygalacturonase, that are important for efficient degradation of xylan. As inferred from 16S rRNA sequences and average nucleotide identity values, the closest relatives of strain DA-C8 are Paenibacillus cisolokensis and P. chitinolyticus. The genomic data have been deposited at the National Center for Biotechnology Information (NCBI) under accession number BMAQ00000000.

Cassava is an important crop, in which root tubers have been known as a staple food in tropical area. Recently, cassava tubers are processed by various methods into numerous products and utilized in various ways. The land area of cassava cultivation has been increasing especially in Southeast Asia. Such a situation is causing an introduction of new cassava diseases and insect pests in the region. In the main part of this chapter, we reviewed the current cassava situation in Vietnam, Cambodia, and Thailand and then illustrated the major cassava diseases and insect pests and their management methods. In addition, we described the international activities developing the cassava agriculture to date, the existing agricultural extension system, and the socioeconomic situation of cassava farmers. Since 2016, we have been conducting SATREPS project to establish a management of invasive cassava diseases and insect pests. At last part chapter, we showed our approach to establish the sustainable agriculture of cassava.

Rice yellow mottle virus (RYMV) is the most important rice virus in Africa. We examined RYMV transmission via soil and water contaminated with RYMV-infected rice plants and by serial cutting with RYMV-contaminated scissors. Transmission of RYMV via dried rice straw kept at 27°C was also examined. The results showed the virus could be transmitted via soil and water, and by scissors. Rice straw that was RYMV-infected was not infective if it was dried and was kept longer than 42 days. By insect transmission experiments and ELISA, long-horned grasshoppers (Conocephalus spp.) were found to be a possible vector of RYMV in Uganda.