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Purpose: A method to control soybean cyst nematode (SCN) is to grow green manure which releases hatching stimulants in the absence of host plants. We hypothesized that biochar may improve the efficacy of this control strategy. Methods: We evaluated the effects of two hatch-stimulating plants (Vigna radiataand Crotalaria spectabilis) in combination with rice husk biochar on the plant growth, SCN density, and soil nematode community. Plants were cultivated in biochar-amended and unamended soils for one month and then incorporated. Soils were collected periodically for chemical properties, SCN, and nematode community assessment. Results: Biochar increased total biomass of V. radiataand C. spectabilissignificantly. For V. radiataalone, the number of SCN juveniles (J2) increased at three weeks and then decreased, but in biochar-amended soil J2 prevailed for three to seven weeks after residue incorporation. In soil incorporated with C. spectabilis, J2 were sustained consistently in biochar-amended soil. Nine weeks after residue incorporation, real-time PCR assays revealed that V. radiatareduced the SCN density by 96% and 91%, and that C. spectabilisreduced the SCN density by 72% and 48% with and without biochar, respectively. Incorporation of green manure in biochar-amended soil increased microbial activity and abundance of omnivorous nematodes, notably Ecumenicusand Aporcelaimellusspecies which were negatively correlated with the SCN density. Specifically, V. radiataincreased the composite and enrichment footprints of nematodes, and omnivore and structure footprints were further increased by combination with biochar. Conclusion: Biochar may improve the efficacy of a green manure-based strategy to control SCN.

ABSTRACTWe aimed to elucidate the effects of decreases in nitrogen (N) inputs on N budgets and soil acidity in N saturated forests in Tokyo, Japan using new analysis and comparison to sampling data collected in the 1990s. Canopy throughfall and soil-percolated water samples were collected from stands of Japanese cedar (Cryptomeria japonica) and Japanese cypress (Chamaecyparis obtusa), where allophanic Andisols have developed. Stream water was sampled at the outlet of the catchment (2.15 ha). The total inorganic N input due to throughfall in the Japanese cypress stand was 104–184 mmolc m−2year−1, which is a marked decrease from sampling results from the 1990s (1991–1992 and 1995). This decrease can be ascribed to a reduction in NOxemission from automobiles. Consequently, nitrate (NO3−) outputs from the catchment decreased when compared with the previous study, although outputs were comparable to N inputs. Based on i) N budgets for the catchment and ii) episodic acidification of stream water, the study site remains N saturated. The degree of decrease in ammonium (NH4+) inputs was larger than NO3−inputs, which resulted in a decrease in acid production due to nitrification or plant uptake and contributed to reduced soil acidification. As a result, the pH values of soil-percolated water in the Japanese cypress stand increased slightly, compared to levels in the 1990s. On the other hand, soil pH values in the Japanese cypress stand were comparable to those in the 1990s due to the large acid-neutralizing capacity of allophanic Andisols.

The lotus root nematode, Hirschmanniella diversaSher (Tylenchida: Pratylenchidae), causes damage to lotus tubers, and thus decreases its economic value. Few studies have elucidated the life cycle of H. diversa, which is essential to optimize control methods. Therefore, we collected H. diversafrom lotus roots and investigated its seasonal population fluctuations, female mating rates, stylet, and body lengths to understand its seasonal occurrence and possibly predict its life cycle. Hirschmanniella diversawere sampled from two lotus paddy fields between 2010 and 2012 in Ibaraki Prefecture, Japan. The occurrence of H. diversaincreased with time peaking around late August and early September, then depreciating thereafter. The seasonal breeding period determined by quantifying the number of H. diversafemales with spermatheca filled with sperm peaked from April until mid-May and decreased subsequently. Furthermore, overwintered adult H. diversafemales laid eggs around June, hence possibly mating between April and May. Second-stage juveniles emerged at the beginning of August and continued developing thereafter. The fourth-stage juveniles or adults overwintered in the lotus roots, weed roots, or soil. The population dynamics of H. diversain lotus roots in paddy fields determined in this study will be useful for development of control strategies.

Chrysanthemum is a very popular flower in Japan and is known to be infected by many soil-borne plant pathogens including nematodes. A nematode survey in six chrysanthemum fields in Okinawa, Japan, found Pratylenchus, Meloidogyne, and Paratylenchus(P. dianthus). The first two genera are known as plant pathogens against chrysanthemum, however, Paratylenchusdianthus has not been reported previously. Chrysanthemum seedlings were grown in pots containing soil infected only with P. dianthusfor two months in 2017 and 2018. The nematicide imicyafos was applied in triplicates to half of the pots (treated) while the other half were left without the nematicide (non-treated). Plant height and dry plant weight of the imicyafos treated plants exceeded those of the control plants. Also, single-photon avalanche diode value of chrysanthemum leaves was higher in imicyafos treated plants than in the non-treated plants at two-month after planting. The results suggest that P. dianthusmay suppress the growth of chrysanthemum. For high-throughput nematode diagnosis, a real-time PCR primer set specific to P. dianthuswas developed and its sensitivity to quantify P. dianthuswas confirmed with a proper calibration curve. The calibration curve was developed in a simplified approach by using serially diluted DNA extracted from individual nematodes.

No nematicide has been registered for controlling plant-parasitic nematodes in sugarcane in Japan. Greenhouse and field experiments were conducted to evaluate the efficacy of the phenylpyrazole insecticide fipronil (0.3–0.45 kg a.i./ha) against the root-lesion nematode, Pratylenchus zeae, which is an important plant-parasitic nematode species associated with sugarcane (Saccharumspp. hybrids) in Okinawa, Japan. Both experiments showed a reduction of P. zeaepopulation densities in sugarcane roots to 27 to 56% of the non-treated control after 7 wk in the greenhouse experiment and 3 months in the field experiment (two trials). In contrast, P. zeaepopulation densities in soil were not reduced by the fipronil treatment. At harvest, sugarcane yields in the field experiment were significantly increased by 6 to 8% in the fipronil treated plots compared to the non-treated control. The data showed that fipronil reduced numbers of P. zeaein sugarcane roots at the early stage of sugarcane seedling growth resulting in increased sugarcane yields. This is the first report of nematicidal activity of fipronil against P. zeaeunder field conditions.

Nematicides, especially 1,3-dichloropropene (D–D), are empirically used to control damage caused by the root-knot nematode Meloidogyne incognita(Kofoid and White) Chitwood in fields of sandy soils in northeast Tokushima, a major sweet potato (Ipomoea batatas(L.) Lam.) producing area. The objective of this study was to elucidate an economic threshold against the nematode damage. Relationship between the nematode density before nematicide use and the degree of nematode damage at harvest was evaluated in nematicide-treated and non-treated plots. There was a significant correlation between the pre-plant nematode densities and nematode damage in the nematicide non-treated plots. Marketable quality of sweet potato decreased in four out of 16 in the non-treated plots, while it did not decrease in all 11 nematicide non-treated plots in which the pre-plant nematode densities were less than 100 second-stage juveniles (J2) equivalent 20 g−1of soil, estimated using a real-time PCR method, suggesting that this density is an economic threshold for damage by root-knot nematodes to sweet potato. The present study demonstrates that nematicides can be avoided in fields containing root-knot nematodes at densities less than the economic threshold.

Abstract This paper focuses on N balance in a paddy field planted with whole crop rice (Oryza sativa cv. Kusahonami). The experiment was conducted with two treatments during two rice-growing seasons: one was fertilized with N (160 kg N ha−1; 16N plot) and the other unfertilized (0N plot); both plots were fertilized with P and K. The N input from precipitation was 15 and 12 kg N ha−1 in 2002 and 2003, respectively. The N input from irrigation water reached as much as 123 and 69 kg N ha−1 in 2002 and 2003, respectively. This was because irrigation water contained higher NO3− concentrations ranging from 4 to 8 mg N l−1. The N uptake by rice plants was the major output: 118 and 240 kg N ha−1 in the 0N and 16N plots in 2002 and 103 and 238 kg N ha−1 in 2003, respectively. N losses by leaching were 4.8–5.3 and 6.5–7.3 kg N ha−1 in 2002 and in 2003, respectively. Laboratory experiments were carried out to estimate the amounts of N2 fixation and denitrification. Amount of N2 fixation was 43 and 0 kg N ha−1 in the 0N and 16N plots, respectively. Denitrification potential was quite high in both the plots, and 90% of the N input through irrigation water was lost through denitrification. Collectively, the total N inputs were relatively large due to irrigation water contaminated with NO3−, but N outflow loading, expressed as leaching−(irrigation water + precipitation + fertilizer), showed large negative values, suggesting that the whole crop rice field might serve as a constructed wetland for decreasing N.

The effect of bensulfuron-methyl (BSM) on a soil microbial community in a model paddy microcosm was studied. Total bacterial numbers in the overlying water and surface soil were monitored for 2 months after the application of BSM at the field rate and a ten-fold field rate. Pentachlorophenol (PCP) was used for comparison. Neither chemical affected the total bacterial numbers remarkably, either in the overlying water or in the surface soil. In contrast, the nitrification potential was significantly suppressed by the BSM application. The bacterial community structure, as evaluated by the denaturing gradient gel electrophoresis (DGGE) of PCR amplification products from bacterial 16S rDNA, was unaffected by the BSM treatments over 8 weeks in the surface soil, compared with the control (no pesticide). In contrast, the surface soil exposed to PCP at a ten-fold field rate showed different patterns from the controls at 4 weeks and 8 weeks after application. The DGGE patterns of the overlying water were much more variable than those of the surface soil in any treatments. Cluster analysis showed that the BSM plots were classified within the same group as the control at 1 week after application and that the BSM and PCP plots from 2 weeks onward after application were grouped differently from the control. Of 22 clones excised from the DGGE gels, 20 clones belonged to the Proteobacteria and two belonged to the Verrucomicrobia. It was considered that the impact of BSM on the overall microbial community (total numbers, community structure of soil) was negligible, although BSM had an impact on some specific functions of the soil microbial community (nitrification) and a part of the community (overlying water).

Abstract. Community structure of the microbiota in rice roots that developed from different nodes and at different growth stages were compared by using PCR (polymerase chain reaction)-RFLP (restriction fragment length polymorphism) analysis of 16S rDNA. This was the first study to have applied a molecular microbial technique for elucidating the rhizospheric microbial succession of rice roots. Rice plants were grown in submerged soil pots, and nodal roots were collected 5 times during the growing period of rice plants. The RFLP fragments digested by four restriction enzymes (Hinf I, Hae III, Sau3A I, and EcoR I) tended to increase along with the growth stage. A marked increase in the RFLP fragments coincided with the development of reduction in the rhizosphere soil. RFLP fragments that were associated with every nodal root irrespective to the sampling date and those specific to the early and late growth stages were identified. Systematic changes in RFLP patterns from higher (younger) nodal roots to lower (older) nodal roots were also observed at each sampling date, which indicated the succession of the microbial community from higher to lower nodal roots. Cluster analysis divided the RFLP patterns of the microbial community associated with nodal roots into four clusters depending on the growth stages of rice plants. The cluster of the early growth stage was further divided into two subclusters of higher and lower nodal roots. The specific RFLP fragments that contributed to the seasonal variation of the microbial community associated with nodal roots as well as those that characterized the aging of nodal roots were clarified by principal component analysis.

AbstractWe investigated the effect of prior colonization of sterile soils by a range of soil microbes on the growth of Ralstonia solanacearumYUIRif43 in an earlier report (Soil Sci. Plant Nutr., 46, 449–459, 2000). Here, we report the effect of prior colonization of tomato roots, the host plant of the pathogen, by soil microbes on succeeding colonization by the pathogen. When the soil microbial community was introduced onto tomato roots, subsequent colonization by YUIRif431ux, a lux-marked mutant of YUIRif43, was remarkably suppressed compared with that in the absence of microorganisms which had previously colonized roots (hereafter referred to as priorcolonists). Soil bacteria, rather than fungi, were mainly responsible for the suppression. Next, the effect of individual microbial strains on the colonization by YUIRif43lux was examined. Some strains did not show any significant suppressive effect on subsequent colonization by YUIRif43Iux, unlike others. All the R. solanacearumstrains used in this study, especially R. solanacearumNPSI (a weakly virulent mutant of YUl), were effective prior colonists against colonization by YUIRif431ux, that is, they markedly suppressed subsequent colonization by YUIRif431ux. Most of the strains that were effective prior colonists in soil in the earlier report were also effective at suppressing the colonization of tomato roots by YUIRif43Iux. YUIRif43lux competed on tomato roots with the strains that did not show previously an effect against colonization by YUIRif431ux, while root colonization by YUlRif43lux was reduced by co-inoculation with the strains that previously showed a colonization effect. These results suggested that soil bacteria were likely to be responsible for the suppression of colonization by YUIRif43lux of tomato roots, and that strains belonging to R. solanacearumamong individual microbial strains might be the most effective antagonists against colonization by YUIRif431ux. Using Tn5-induced derivatives of a strain, Pseudomonas fluorescensMelRC2, one of the most effective priorcolonists, the contribution of motility, antibiosis, rhizosphere competence to the suppressiveness of P. fluorescensMelRC2against colonization by YUIRif43lux were examined.

Abstract:  Microbial populations of the earthworm Eisenia foetida, their casts and farmyard manure (FYM) in which E. foetida had lived were enumerated with the dilution plate method using several media. The microbial community of earthworms appeared to be similar to that of the FYM in which they had lived, while that of earthworms starved in distilled water for 24 h greatly differed from that of the FYM. The community of the starved earthworms consisted exclusively (more than 90%) of fast growers, which were defined as those that formed their colonies within 2 days, unlike that of the FYM (10–30%). About 30 isolates were randomly chosen and some physiological properties were examined. The community of starved earthworms consisted exclusively of Gram-negative, oxidase-positive, fermentative bacteria, whereas bacteria from the FYM were much more diverse and there were no fermentative bacteria in the FYM. The predominant bacteria species of the starved earthworms were identified as Aeromonas hydrophila and considered to be indigenous to the earthworm E. foetida.

AbstractThe effect of prior colonization of a sterile loam soil and a sterile clay loam soil by individual soil bacteria on the subsequent growth of a bacterial wilt pathogen Ralstonia solanacearumYU1Rif43 (tRNA type III: Seal et al. 1992: Appl. Environ. Microbiol., 58, 3759–3761) was investigated. Various strains, belonging to the same type, the same species, the same genus, Gram-negative, Grampositive, or fungi, were used. The degree of suppression of the growth of R. solanacearumYU1Rif43 was markedly different depending on the species that had previously colonized the soil, hereafter referred to as priorcolonists. All the strains belonging to R. solanacearumtype III suppressed the growth of R. solanacearumYU1Rif43 markedly, while strains of R. solanacearumtype I and type II showed a moderate suppressive effect on R. solanacearumYU1Rif43. The suppressive effect of the strains belonging to species other than R. solanacearum, including fungal strains, was relatively limited, or some strains did not show any suppressive effect. The production of bacteriocin did not appear to be related to the strong suppressive effect of the R. solanacearumtype III strains. Possible mechanisms for the suppressive effect of priorcolonists on R. solanacearumYU1Rif43 are discussed in relation to nutrients and physical sites in soil available for growth.

AbstractThe effects of fumigation with sodium methyl dithiocarbamate (metam sodium) on the microbial community structure and function in 2 soils were investigated using a variety of techniques. In both soils ca. 50% and 90% of the populations of total and culturable bacteria, respectively, were killed by fumigation, with recovery to levels prevailing in control soils 26 d after cessation of fumigation. The size of the ammonium and nitrite oxidiser populations was reduced by up to 4 orders of magnitude by fumigation, with the latter showing a slight recovery 105 d later. There were substantial changes in the C-utilisation (Biolog GN) profiles in the fumigated soils even 105 d later. The number and pattern of amplified 16S ribosomal DNA restriction analysis (ARDRA) fragments was changed by fumigation, and there was a shift in the %G+C profile toward a greater proportion of lower %G+C classes in treated soils. It appeared that DNA released from killed cells remained for some time after fumigation, and masked the apparent community DNA profiles. This study demonstrates that the effects of fumigation on the soil microbial community structure and function were pronounced and for some parameters very persistent. However, the effects on broad-scale properties such as total or culturable bacterial numbers were less enduring.

AbstractThe surface of micro crustaceans collected from the overlying water of a submerged paddy soil was observed in detail by scanning electron microscopy. Epibiotic bacteria were less diverse compared to free-living bacteria in the overlying water. Specific bacteria appeared to preferentially colonize certain parts of microcrustaceans. The epibiotic bacteria, the sites they colonized and their density depended on the kinds of microcrustaceans (Moina macrocopa, Cyclopidae, Cyprettasp., and Tanycyprissp.). These results suggested the presence of significant associations in the overlying water between specific groups of bacteria and microcrustaceans.

AbstractAgricultural practices, such as the application of fertilizers and pesticides, differently influenced the seasonal variations of respective microcrustaceans. Microbial flora on the surface of microcrustaceans was also affected by the practices and varied depending on the kinds of microcrustaceans and the date of collection. The microbial flora on the surface of microcrustaceans was very simple, often comprised of a few types of bacteria based on the morphological characters, and their colonization sites were restricted. These results suggested the preferential colonization of selective bacteria on specific sites of host microcrustaceans. This is the first report showing the close association of microorganisms with micro crustaceans living in paddy fields.

AbstractBacteria responsible for the suppression of Fusarium oxysporumf. sp. raphanion radish roots grown in so-called disease-suppressive (S) soils were isolated and characterized. About six hundred strains of bacteria, actinomycetes, Gram-negative bacteria, fluorescent pseudomonads, and antagonistic microorganisms were isolated from the roots grown in S-soils and so-called conducive (C) soils. Isolates were selected on the basis of in vitro antibiosis to the pathogen on potato dextrose agar. Effective isolates were obtained with a relatively high frequency from the roots grown in S-soils compared with C-soils. One (MRT11) of the isolates from a S-soil, Miura soil, identified as Pseudomonas cepacia, enabled to recover the suppressiveness to the pathogen on the roots to almost the same degree as in the case of natural roots grown in S-soils when surface-sterilized roots were dipped in a suspension of the isolate. This result suggested that MRT11 was an isolate responsible for the suppression of the pathogen on roots grown in Miura soil. Roots grown in C-soils became suppressive to the pathogen when seedlings were dipped in a MRT11 culture, and MRT11 was able to survive on roots grown in both S- and C-soils.

AbstractSuppression mechanisms involved in so-called suppressive soils to Fusarium-wilt of radish, caused by Fusarium oxysporumf. sp. raphani, were investigated. Growth and survival of the pathogen in nonrhizosphere soils were not different between so-called disease-suppressive (S) and conducive (C) soils. The growth of the pathogen on radish roots grown in S-soils was suppressed compared with C-soils, which suggested that S-soils displayed a greater degree of fungistasis than C-soils. The suppressiveness to the pathogen was removed by surface-sterilization of roots. It was therefore considered that the suppressiveness to the pathogen was due to the microorganisms colonizing the roots grown in S-soils. Estimation of microbial populations on roots suggested that the suppression may be due to antagonistic microorganisms.