Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain.

IMPORTANCE: DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.

Characteristics of the Complete Genome of Ageratum Yellow Vein China Virus Infecting Sonchus oleraceus.

Sonchus (Sonchus oleraceus) originated from Europe and is now cultivated worldwide. The wild resources of sonchus are very abundant, and it has rich nutritional and medicinal value. In this study, 15 sonchus samples with typical symptoms showing leaf curling, vein thickening, and enations were collected from Guigang and Baise City of Guangxi, China. Diseased sonchus were identified by PCR detection, whole genome sequence amplification, and phylogenetic and recombination analysis. The results showed that all the samples were confirmed infected by begomoviruses, and three full-length viral genomes were obtained from 15 sonchus, named GG7-13, GG8-6, and BS63-5. The full genome lengths were 2,584, 2,735, and 2,746 nt, respectively. The nucleotide identities among the three isolates ranged from 92.67 to 99.93%. All of them shared the highest identities (greater than 91.69%) with other isolates of ageratum yellow vein China virus (AYVCNV) (available on GenBank). According to the guidelines of classification of begomoviruses, the virus isolates obtained in this study are different isolates of AYVCNV; a phylogenetic tree analysis showed that these isolates formed a large branch with three other Guangxi isolates of AYVCNV, indicating their close evolution. The genome structures of GG8-6 and BS63-5 are consistent with the monopartite genome virus of the begomoviruses, and both have six open reading frames (ORFs), while GG7-13 has a 151-nt deletion between C2 and C3, resulting in a mutant strain of only five ORFs. This study is the first report on S. oleraceus infected by ageratum yellow vein China virus.

[Novel DNA-beta associated molecules produced by sequence recombination and deletion of cotton leaf curl Multan virus complex].

OBJECTIVE: Cotton leaf curl disease (CLCuD) is a major constraint to cotton production, causing great economic losses in Pakistan and India. In China, CLCuD has been discovered in the field of Nanning, GuangXi. To better understand this disease, we sequenced the virus-associated small DNA molecules. METHODS: We purified total DNA from cotton and okra plants exhibiting leaf curl symptoms; PCR amplified and sequenced CLCuMV satellite DNA (DNAbeta) -related small DNA molecules. RESULTS: We identified 2 novel recombinant DNA molecules with 1384 nucleotides in cotton and 754 nucleotides in okra. The 1384 nt molecule contains partial DNA-A and DNAbeta of CLCuMV GX1. It includes the intergenic region, adjacent AV2 and AC1 coding sequences, and reverse complementary AC3 of DNA-A and A-rich region of DNAbeta. Common nucleotides were found around the junction points of DNA-A and DNAbeta sequences, suggesting they were the sites of recombination. Comparison with previous reported CLCuMV recombinants produced in lab showed that the intergenic region of DNA-A and A-rich region of DNAbeta were conserved on the recombination process. The 754 nt molecule was produced by deletion of CLCuMV DNAbeta in the C1 open reading frame and A-rich region. CONCLUSION: We identified a novel recombinant molecule originated from CLCuMV DNA-A and DNAbeta and a small defective molecule of DNAbeta. This is the first report of sequence recombination and deletion of CLCuMV in China, which may be helpful to understand the CLCuMV evolution and host adaptation.