Deyong Zhang

Selection of reference genes for RT-qPCR analysis in a predatory biological control agent, Coleomegilla maculata (Coleoptera: Coccinellidae)

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, RefFinder, a comprehensive platform integrating all the above-mentioned algorithms, ranked the overall stability of these candidate genes. As a result, various sets of suitable reference genes were recommended specifically for experiments involving different tissues, developmental stages, sex, and C. maculate larvae treated with dietary double stranded RNA. This study represents the critical first step to establish a standardized RT-qPCR protocol for the functional genomics research in a ladybeetle C. maculate. Furthermore, it lays the foundation for conducting ecological risk assessment of RNAi-based gene silencing biotechnologies on non-target organisms; in this case, a key predatory biological control agent.

Genome Sequence of Pyrethroid-Degrading Bacterium Rhodopseudomonas palustris Strain JSC-3b

ABSTRACT Rhodopseudomonas palustris strain JSC-3b is a facultative, thermophilic bacterium, which was isolated from water in a canal adjacent to a vegetable field. Strain JSC-3b biodegrades several varieties of pyrethroid residues effectively through cometabolic pathways. Here, we present the genome sequence of this biodegrader.

Isolation of Rhp-PSP, a member of YER057c/YjgF/UK114 protein family with antiviral properties, from the photosynthetic bacterium Rhodopseudomonas palustris strain JSC-3b

Rhodopseudomonas palustris strain JSC-3b isolated from a water canal adjacent to a vegetable field produces a protein that was purified by bioactivity-guided fractionation based on ammonium sulfate precipitation, ion-exchange absorption and size exclusion. The protein was further identified as an endoribonuclease L-PSP (Liver-Perchloric acid-soluble protein) by shotgun mass spectrometry analysis and gene identification, and it is member of YER057c/YjgF/UK114 protein family. Herein, this protein is designated Rhp-PSP. Rhp-PSP exhibited significant inhibitory activities against tobacco mosaic virus (TMV) in vivo and in vitro. To our knowledge, this represents the first report on the antiviral activity of a protein of the YER057c/YjgF/UK114 family and also the first antiviral protein isolated from R. palustris. Our research provides insight into the potential of photosynthetic bacterial resources in biological control of plant virus diseases and sustainable agriculture.

Pumpkin powdery mildew disease severity influences the fungal diversity of the phyllosphere

Phyllosphere microbiota play a crucial role in plant-environment interactions and their microbial community and function are influenced by biotic and abiotic factors. However, there is little research on how pathogens affect the microbial community of phyllosphere fungi. In this study, we collected 16 pumpkin (Cucurbita moschata) leaf samples which exhibited powdery mildew disease, with a severity ranging from L1 (least severe) to L4 (most severe). The fungal community structure and diversity was examined by Illumina MiSeq sequencing of the internal transcribed spacer (ITS) region of ribosomal RNA genes. The results showed that the fungal communities were dominated by members of the Basidiomycota and Ascomycota. The Podosphaera was the most dominant genus on these infected leaves, which was the key pathogen responsible for the pumpkin powdery mildew. The abundance of Ascomycota and Podosphaera increased as disease severity increased from L1 to L4, and was significantly higher at disease severity L4 (P < 0.05). The richness and diversity of the fungal community increased from L1 to L2, and then declined from L2 to L4, likely due to the biotic pressure (i.e., symbiotic and competitive stresses among microbial species) at disease severity L4. Our results could give new perspectives on the changes of the leaf microbiome at different pumpkin powdery mildew disease severity.

Transmission Efficiency, Preference and Behavior of Bemisia tabaci MEAM1 and MED under the Influence of Tomato Chlorosis Virus

Tomato chlorosis virus (ToCV, genus Crinivirus, family Closteroviridae) is an economically important virus in more than 20 countries. In China, ToCV was first detected in 2013 and has already spread throughout the country. ToCV is transmitted in a semi-persistent manner by the whitefly, Bemisia tabaci, but not seed. In the past two decades, the most invasive MEAM1 and MED have replaced the indigenous B. tabaci in China, and currently MED is the most dominant cryptic species. To better understand the prevalence of ToCV with their vectors, we tested the hypothesis that the rapid spread of ToCV in China is closely related to the dominance of MED. ToCV acquisition and accumulation rate following transmission was significantly higher by MED than MEAM1. In addition, ToCV persisted for more than 4 days in MED but only 2 days in MEAM1. Viruliferous MED preferred non-infected over virus-infected plants, although MED performed better on infected than on non-infected plants. Our combined results support the initial hypothesis that the rapid spread of ToCV is associated with the spread of B. tabaci MED in China.

Photosynthetic bacterium Rhodopseudomonas palustris GJ-22 induces systemic resistance against viruses

Photosynthetic bacteria (PSB) have been extensively used in agriculture to promote plant growth and to improve crop quality. Their potential application in plant disease management, however, is largely overlooked. In this study, the PSB strain Rhodopseudomonas palustris GJ‐22 was investigated for its ability to induce resistance against a plant virus while promoting plant growth. In the field, a foliar spray of GJ‐22 suspension protected tobacco plants against tobacco mosaic virus (TMV). Under axenic conditions, GJ‐22 colonized the plant phyllosphere and induced resistance against TMV. Additionally, GJ‐22 produced two phytohormones, indole‐3‐acetic acid and 5‐aminolevulinic acid, which promote growth and germination in tobacco. Furthermore, GJ‐22‐inoculated plants elevated their immune response under subsequent TMV infection. This research may give rise to a novel biological agent with a dual function in disease management while promoting plant growth.

Development and Evaluation of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Tylenchulus semipenetrans Using DNA Extracted from Soil

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at 63°C for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was 10−2 J2/0.5 g of soil, which was 10 times more sensitive than conventional PCR (10−1 J2/0.5 g of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

Biodegradation of fenpropathrin by a novel Rhodopseudomonas sp. strain PSB07-8

The aim of our study was to isolate a fenpropathrin-degrading bacterial strain and to determine its effectiveness in bioremediating fenpropathrin-contaminated soil in the laboratory. We isolated a bacterial strain from soils and named it as PSB07-8, and showed that this organism was able to degrade pyrethroid pesticide fenpropathrin. The organism was identified as a strain of Rhodopseudomonassp.The strain PSB07-8 degraded fenpropathrin in a co-metabolic way, and the optimal condition of degradation was at temperature of 30°C and pH value of 7. Moreover, the strain PSB07-8 could effectively degrade cypermethrin and bipthenthrin, but only slightly for deltemethrin, cyfluthrin and esfenralerate. We found that the degradation rate was negatively correlated with the fenpropathrin concentration in a certain range. In addition, our soil degradation test showed that in sterilised soils with 20 mg/l or 40 mg/l fenpropathrin, the strain PSB07-8 could complete 100% or achieve 44.7% of fenpropathrin degradation in 15 days. To our best knowledge, this is the first report of a strain of genus Rhodopseudomonas with an ability of degrading fenpropathrin in soil.

Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385 , from Rhodopseudomonas palustris PSB-S

Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30–46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l−1 and 0.918 ± 0.025 U·µg−1, respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

Detection and epidemic dynamic of ToCV and CCYV with Bemisia tabaci and weed in Hainan of China

BackgroundIn recent years, two of the crinivirus, Tomato chlorosis virus (ToCV) and Cucurbit chlorotic yellows virus (CCYV) have gained increasing attention due to their rapid spread and devastating impacts on vegetable production worldwide. Both of these viruses are transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), in a semi-persistent manner. Up to now, there is still lack of report in Hainan, the south of China.MethodsWe used observational and experimental methods to explore the prevalence and incidence dynamic of CCYV and ToCV transmitted by whiteflies in Hainan of China.ResultsIn 2016, the chlorosis symptom was observed in the tomato and cucumber plants with a large number of B. tabaci on the infected leaves in Hainan, China, with the incidence rate of 69.8% and 62.6% on tomato and cucumber, respectively. Based on molecular identification, Q biotype was determined with a viruliferous rate of 65.0% and 55.0% on the tomato and cucumber plants, respectively. The weed, Alternanthera philoxeroides near the tomato and cucumber was co-infected by the two viruses. Furthermore, incidence dynamic of ToCV and CCYV showed a close relationship with the weed, Alternanthera philoxeroides, which is widely distributed in Hainan.ConclusionOur results firstly reveal that the weed, A. philoxeroides is infected by both ToCV and CCYV. Besides, whiteflies showed a high viruliferous rate of ToCV and CCYV. Hainan is an extremely important vegetable production and seed breeding center in China. If the whitefly can carry these two viruses concurrently, co-infection in their mutual host plants can lead to devastating losses in the near future.