Study on PCR rapid molecular detection technique of Meloidogyne vitis

Meloidogyne vitis is a new root-knot nematode parasitic on grape root in Yunnan Province,China.In order to establish a rapid,reliable and specific molecular detection method for M.vitis,the species-specific primers were designed with rDNA-ITS(ribosomal DNA internal transcribed spacer)gene fragment as the target.The reaction system was optimized and the reliability,specificity and sensitivity of primer were testified,therefore,a rapid PCR detection method for M.vitis was established.The result showed that the optimal annealing temperature of the primers was 53℃,which was suitable for the detection of different life stages of M.vitis.Specificity test showed that the specific fragment size of 174 bp was obtained from M.vitis,but other five non-target nematodes did not have any amplification bands,thus effectively distinguish M.vitis and the other five species,and could specifically detect the M.vitis from mixed populations.Sensitivity test showed that this PCR technique could detect the DNA of a single second-stage juvenile(J_(2))and 10^(-4)female.Futhermore,this PCR technique could be used to detect directly M.vitis from soil samples.The rapid,sensitive and specific PCR molecular detection technique could be used for the direct identification of a single J_(2)of M.vitis and the detection of M.vitis in mixed nematode populations and the detection of two J_(2)s or one male in 0.5 g soil samples,which will provide technical support for the investigation of the occurrence and damage of M.vitis and the formulation of efficient green co ntrol strategies.

Detection and Identification of Sugarcane Pokkah Boeng Pathogens of Main Sugarcane Varieties in Yunnan Province

[Objectives]This study was conducted to identify the pathogen species and dominant species of sugarcane pokkah boeng in main varieties in Yunnan sugarcane areas,so as to promote the healthy and sustainable development of sugarcane industry.[Methods]Specific primers Fv-F3/Fv-R3 and Fp-F4/Fp-R4 were designed based on the ribosomal DNA non-internal transcribed spacer(rDNA-ITS)gene sequences of Fusarium verticillioides and Fusarium proliferarum,the main pathogens of sugarcane pokkah boeng,and 117 typical sugarcane pokkah boeng samples collected from main varieties in different sugarcane areas of Yunnan Province were detected and analyzed by PCR.[Results]Among the 117 sugarcane pokkah boeng samples,112 samples were detected with F.verticillioides with a positive detection rate of 95.7%;103 samples were detected with F.proliferarum with a positive detection rate of 88%;103 samples were infected by F.verticillioides+F.proliferarum,and the compound infection rate was 88%;and the two pathogens were not detected in 5 samples,which might be sugarcane pokkah boeng caused by other species.PCR amplification products of 23 F.verticillioides positive samples and 19 F.proliferarum positive samples from different sugarcane varieties in different sugarcane areas were sequenced.The BLAST alignment results showed that the sequences of the 23 amplification products of F.verticillioides shared 99.45%-100.00%homology with F.verticillioides(GenBank accession number:KU508286),and the sequences of the 19 amplification products of F.proliferarum shared 99.26%-100.00%homology with the sequence of F.proliferarum(GenBank accession number:MK252904).Part of the F.verticillioids and 11 F.proliferarum sequences were selected to construct a phylogenetic tree,and the phylogenetic analysis showed that they belonged to the F.verticillioids group and F.proliferarum group,respectively.The results showed that F.verticillioides and F.proliferarum were the main pathogens causing sugarcane pokkah boeng of the main sugarcane varieties in Yunnan,and there was a common phenomenon of compound infection.F.verticillioides was the dominant species in Pu er,Lincang,Honghe and Yuxi sugarcane areas,but the detection rate of F.proliferarum was also high,and there were other species.In the future,the discovery of resistant germplasm resources and breeding of resistant varieties should be carried out aiming at these two pathogens of sugarcane pokkah boeng.[Conclusions]The study provides technical support for rapid identification of sugarcane pokkah boeng pathogens,and scientific basis for breeding resistant varieties and scientific disease prevention and control.

Study on the Genetic Transformation Conditions of Begonia wallichiana L.with Leaf Disc Method and Corresponding Identification Techniques

[Objectives]This study was conducted to improve the Agrobacterium-mediated genetic transformation system of Begonia wallichiana.[Methods]With sterilized tube seedling leaves as the recipient material and GFP as the reporter gene,optimization experiments were carried out in terms of infection time and method,co-cultivation time and method,and PCR detection technology.[Results]The transformation effect was better under the conditions of shaking Agrobacterium liquid,infection time of 1-2 h,and co-cultivation on sterilized filter paper for 2 d.After co-cultivation,the recipient material was first subjected to recovery culture,and then used for Hyg gradient screening,which was conducive to obtaining resistant transformants.The designed specific PCR detection technology could quickly identify false positives in resistant regenerated plants,and the proportion of transgenic plants was 16.7%.[Conclusions]The research results provide a new technical reference for the genetic transformation of ornamental plants.

Genetic Diversity of Fusarium solani f. sp. cucurbitae, the Causal Root and Crown Rot of Cucurbits (Melon) by Using Molecular Markers and Control

Detection of F. solani f. sp. cucurbitae causal agent of the crown and root rot disease of melon race 1, race 2 is difficult. It is based only on morphological characteristic. In this study, forty isolates identified as Fusarium solani based on morphological characterization, F. solani was one of the most frequently isolated species. Molecular identification of these isolates by PCR technique using species-specific primer, indicated that thirty-two isolates, amplified product 580 bp (race 1) and two isolate amplified product 580 bp (race 2), while six isolates were not amplified with primer of both races. Production of Trichothecenes (T2-toxen, DON.) by Fusarium solani was conducted on isolates confirmed as belonging in the F. solani by PCR. The results indicated that the presence of Tri5, Tri13 genes is coding the ability of synthesis mycotoxin. In vitro, the results indicated that NPs (AgNPs, MgNPs) and chemical (Phylex) possess the antifungal properties against at various level. Treatment with (AgNPs 150 ppm, MgNPs 2%, 3% ppm) and 3% Phylex resulted in maximum inhabitation of F. solani . In vivo, five characters (height plant, hoot ant root fresh and dry weight) were measured based on the greenhouse, field experimental results. Treatment with (AgNPs, MgNPs) and Phylex had higher measured parameters than positive control.

Establishment and evaluation based of a RIG-G gene detection system by TaqMan-MGB probe real-time PCR

Objective To establish a Taq Man-MGB fluorescent probe characterized real-time polymerase chain reaction(q PCR)method for detecting retinoic acid induced genes G(RIG-G)in human acute promyelocytic leukemia(M3).Analyze RIG-G expression levels in peripheral blood of both normal persons and M3 patients and

Strongyloides stercoralis prevalence and diagnostics in Vientiane, Lao People’s Democratic Republic

Background:Despite the high prevalence of strongyloidiasis in the Laotian population,Laotian hospitals still lack diagnostic capacity to appropriately diagnose Strongyloides stercoralis infections.This cross-sectional hospital-based study was conducted to assess the prevalence of Strongyloides stercoralis infection among hospitalized patients treated at Mahosot Hospital,the primary reference hospital of Lao People’s Democratic Republic(Lao PDR),and to validate feasible methods for diagnosing S.stercoralis infection at hospital’s laboratory.Methods:Between September and December 2018,stool samples of 104 inpatients were investigated for S.stercoralis infection by wet smear,Baermann technique,Koga Agar plate culture(KAPC),and real-time detection polymerase chain reaction(RTD-PCR)at the Infectious Diseases Ward of the Mahosot Hospital in Vientiane.The sensitivity,the specificity,the negative predictive value(NPV)of each diagnostic test,as well as their combination(s)was calculated using a composite reference standard(CRS).The correlation of the different test methods was assessed by chi-square or Fisher’s exact test.Cohen’s kappa coefficient was used to assess the diagnostic agreement of the different test methods.Results:The overall prevalence of S.stercoralis infections among the study population was 33.4%.The cumulative infection prevalence statistically significantly increased from the lowest age group of 40 years and below(22.4%),to the medium(40.0%)and to the oldest age group of 61 year and above(72.7%)(P=0.003).The cumulative infection prevalence of CRS was considerably higher in male(40.4%)compared to female patients(28.1%),but not statistically different(P=0.184).The diagnostic sensitivity of Baermann technique,KAPC,RTD-PCR,and the combination of Baermann technique and KAPC were 60.0,60.0,74.3,and 77.1%,respectively.Only 13 patients(37.1%)of the total 35 S.stercoralis patients diagnosed with any technique had a simultaneously positive diagnostic test with Baermann,KAPC and RTD-PCR.Conclusions:We identified Baermann technique and KAPC to be currently the most feasible and implementable standard methods for diagnosing S.stercoralis at a hospital setting such as Mahosot Hospital and provincial and district hospitals in Lao PDR and other low-and middle income countries in Southeast Asia.Trial registration:This study was approved by the National Ethics Committee for Health Research in Lao PDR(reference no.083/NECHR)and by the Ethics Committee Northwest and Central Switzerland(reference no.2018–00594).