T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization.

Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

Effect of primary and secondary Fasciola gigantica infection on specific IgG responses, hepatic enzyme levels and weight gain in buffaloes.

Buffaloes, as highly susceptible definitive hosts of Fasciola gigantica, suffer from a high infection rate of fasciolosis, which causes enormous economic losses. Repeat infection is responsible for this high rate; thus, elucidating the protective immunity mechanism in repeat infection is decisive in fasciolosis prevention. Herein, a secondary experimental infection model was established to preliminarily reveal the protective immunity that occurs in repeat infection. In brief, animals were assigned to three groups: group A (uninfected control), group B (primary infection) and group C (secondary infection). Buffaloes were autopsied 20 weeks post-infection for measurements of the recovered flukes and hepatic examination. In addition, the detection of specific antibody (IgG) responses to F. gigantica excretory-secretory product (FgESP) throughout the whole period and weight gain throughout the first 4 months as a percentage (%) of the starting weight were also determined. The serum hepatic enzyme gamma glutathione transferase (GGT) levels were monitored to assess hepatic damage throughout the study period. Infection establishment was compared between group B and group C. Similar specific IgG patterns were observed between group B and group C, and hepatic damage was more severe in group C than group B. Significant differences in weight gain as a percentage of the start weight were observed between group A and group B at the 3rd and 4th months postprimary infection, while significant differences were not observed between group A and group C or group B and group C. Our results suggest that challenge infection cannot induce resistance against F. gigantica in buffaloes, which is consistent with the protective immunity against Fasciola hepatica reinfection observed in sheep and goats.

First report of Meloidogyne arenaria on roots of Grona triflora in Guangdong Province, China.

Grona triflora (Desmodium triflorum), a perennial herbaceous legume, is widely distributed in southern China. G. triflora has antipyretic, antiseptic and expectorant properties and can therefore be used as a phytomedicine (Ghosal et al. 1973). In July 2020, roots of G. triflora were investigated for nodules and rhizobia collection at the Shibaluohan Mountain Forest Park of Guangzhou. Root galls induced by a root-knot nematode were observed on 90% of the G. triflora samples (in a 200 m2 plot) and the infested plants had yellow, small and withered leaves compared with the healthy ones. The galls number on a G. triflora root ranged from 43 to 92 and the population densities of second stage juveniles (J2s) ranged from 573 to 894 per 100 cm3 soil surrounding the plant. The female perineal patterns showed a low dorsal arch, with lateral field marked by forked and broken striae, no punctate markings between the anus and tail terminus, which matched with the description of Meloidogyne arenaria (Hartman and Sasser 1985). The J2s had the following morphometric characters (n = 15): body length = 501.05 ± 23.71 µm; body width = 17.14 ± 1.23 µm; DGO = 3.13 ± 0.27 µm; stylet length = 12.97 ± 1.38 µm; tail length = 58.02 ± 4.77 µm; hyaline tail terminus = 10.08 ± 0.65 µm. DNA from four female nematodes was isolated for PCR-based diagnostic analyses. A fragment between the COII and LrRNA genes of the mitochondrial DNA was amplified with primers C2F3/1108 (Powers and Harris 1993). In addition, a 28S ribosomal DNA D2/D3 region was amplified with primers MF/MR (Hu et al. 2011). The amplicons were sequenced (GenBank No. MW315989 and MW307358). Nucleotide BLAST results indicated that both sequences show 100% identity with corresponding M. arenaria sequences of isolates from various countries such as Brazil, China, Myanmar and Vietnam (e.g., MK033428, JQ446377, KY293688 and MK026624). For further confirmation, sequence characterized amplified region (SCAR) PCR was employed using the M. arenaria specific primers Far/Rar (Zijlstra et al. 2000). The amplicon was also sequenced (GenBank No. MW315990). The Nucleotide BLAST results showed >99% identity with M. arenaria isolates from Indonesia and Argentina (KP234264, KP253748 and MK015624). Greenhouse tests were conducted to analyze the capacity of M. arenaria to induce galls on G. triflora roots. The G. triflora seeds were collected from the sampling plot and germinated on 0.8% (W/V) agar plates. Then the seedlings were planted in 14 cm deep and 15 cm diam pots filled with sterilized soil from sampling plot. Every seedling was inoculated with 2,000 J2s (n = 15) and plants without J2s were used as a control. Two months later, galls were observed for inoculated roots while no galls were formed on roots of control plants. An average of 13,300 J2s and eggs of M. arenaria (reproduction factor = 6.65) were recovered from the root. Stanton and Rizo (1988) found that G. triflora was susceptible to M. javanica in Australia, and Ogbuji (1978) reported that a population of M. incognita reproduced on roots of G. triflora in Nigeria after artificial inoculation. To our knowledge, this is the first report on G. triflora parasitized by M. arenaria in Guangdong province. M. arenaria has potential to infest local, economically important plants like citrus, pomelo, sugarcane, maize and peanut. As G. triflora is widely distributed in southern China, there is the risk of spreading M. arenaria into agricultural and horticultural systems, that will cause yield loss and economic impacts.

Nitrogen deposition raises temperature sensitivity of soil organic matter decomposition in subtropical forest.

Subtropical ecosystems are strongly affected by nitrogen (N) deposition, impacting soil organic matter (SOM) availability and stocks. Here we aimed to reveal the effects of N deposition on i) the structure and functioning of microbial communities and ii) the temperature sensitivity (Q10) of SOM decomposition. Phosphorus (P) limited evergreen forest in Guangdong Province, southeastern China, was selected, and N deposition (factor level: N (100 kg N ha-1 y-1 (NH4NO3)) and control (water), arranged into randomized complete block design (n = 3)) was performed during 2.5 y. After that soils from 0 to 20 cm were collected, analyzed for the set of parameters and incubated at 15, and 25, and 35 °C for 112 days. N deposition increased the microbial biomass N and the content of fungal and Gram-positive bacterial biomarkers; activities of beta-glucosidase (BG) and acid phosphatase (ACP) also increased showing the intensification of SOM decomposition. The Q10 of SOM decomposition under N deposition was 1.66 and increased by 1.4 times than under control. Xylosidase (BX), BG, and ACP activities increased with temperature under N but decreased with the incubation duration, indicating either low production and/or decomposition of enzymes. Activities of polyphenol-(PPO) and peroxidases (POD) were higher under N than in the control soil and were constant during the incubation showing the intensification of recalcitrant SOM decomposition. At the early incubation stage (10 days), the increase of Q10 of CO2 efflux was explained by the activities of BX, BQ, ACP, and POD and the quality of the available dissolved organic matter pool. At the later incubation stages (112 days), the drop of Q10 of CO2 efflux was due to the depletion of the labile organic substances and the shift of microbial community structure to K-strategists. Thus, N deposition decoupled the effects of extracellular enzyme activities from microbial community structure on Q10 of SOM decomposition in the subtropical forest soil.

The 22nd Chromatography Component of the Fasciola gigantica Excretory-Secretory Products Decreased the Proliferation of Peripheral Blood Mononuclear Cells from Buffalo.

The 22nd chromatography component (F22) of the Fasciola gigantica excretory-secretory products (FgESP) shows better diagnostic value than the FgESP, and diagnostic methods based on F22 have also been established. Thus, exploring its immunomodulatory function and potential as a molecular vaccine candidate is attractive. In the present study, the effect of F22 on the mitogen-induced proliferation of buffalo peripheral blood mononuclear cells (PBMCs) in the innate immune response was preliminarily studied using the FgESP as a control. PBMCs were incubated with concanavalin A (ConA) and phytohemagglutinin (PHA) at optimal (1 µg/well) or suboptimal (0.25 µg/well) doses coupled with FgESP and F22 at different doses (1-16 µg/well). Cell proliferation was then assessed by microenzyme reaction colorimetry (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay). In addition, the components of F22 were also explored by mass spectrometry and then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to infer their functions. The results indicated that FgESP decreased the proliferation of PBMCs stimulated with ConA and PHA at specific doses, whereas F22 significantly decreased the proliferation of PBMCs stimulated with ConA and PHA at both optimal and suboptimal doses (p < 0.05). Two hundred and sixteen proteins were identified in F22, and these included 86 proteins that could be assigned to more than one pathway and some with robust immunomodulatory ability. Further studies should be performed to investigate the immunomodulatory function of F22 in the adaptive immune response, and the components of F22 can be further studied as potential vaccine candidate molecules.

Microbial adaptation to long-term N supply prevents large responses in N dynamics and N losses of a subtropical forest.

Atmospherically-deposited nitrogen (N) can stimulate complex soil N metabolisms and accumulations over time. Whether long-term (decadal) N deposition effects on soil N transformations and functional microbes differ from the short-term (annual) effects has rarely been assessed. Here we conducted a laboratory 15N tracing study with soil samples from a short-term (one year) N addition site and a long-term (12 years) site in a subtropical forest. The effects of simulated N deposition on soil N2O emissions, N transformation rates and microbial nitrifying and denitrifying genes were determined. Our results showed that: (1) long-term N addition did not change soil N2O fluxes significantly in comparison to the short-term N addition. Denitrification, heterotrophic nitrification and autotrophic nitrification contributed 53%, 28% and 18% to total N2O emissions, respectively. (2) Autotrophic nitrification was the dominant N transformation process, except for the high-N treatment at the long-term site. The magnitude of soil N transformation rates was significantly different among N addition treatments but not between short- and long-term N addition sites. However, long-term N addition changed the responses of specific N transformation rates to N addition markedly, especially for the rates of nitrification, organic N mineralization to NH4+, NO3- immobilization and dissimilatory NO3- reduction to NH4+ (DNRA). (3) Responses of ammonia oxidizing archaea and bacteria (AOA and AOB) were more variable than those of denitrifying N2O-producers (nirK) and denitrifying N2O-reducers (nosZ), particularly at the long-term site. (4) The close correlations among N2O flux, functional genes and soil properties observed at the short-term site were weakened at the long-term site, posing a decreased risk for N losses in the acid subtropical forest soil. There is evidence for an adaptation of functional microbial communities to the prevailing soil conditions and in response to long-term natural and anthropogenic N depositions.