Research on the Pathogen of Rice Sheath Blight(Rhizoctonia solani Kühn)and Its Pathogenicity in Sichuan Regions

[ Objective] The paper was to study the cultural characteristic of the pathogen of Rice Sheath Blight （ Rhizoctonia solani Kuhn） and its pathogenicity in Sichuan regions. [ Method] The samples of rice sheath blight collected from six main rice planting areas in Sichuan regions were separated. The separated pathogen of rice sheath blight was cultured on PDA medium, and its cultural characteristic was recorded. Meanwhile, the pathogenicity of the obtained 23 strains was determined. [ Result] The growth rates among different strains had significant difference. According to the growth rate, only one strain belonged to medium strain（colony diameter： 40 mm≤（Ф≤60 mm）, the rest were all slow-type strains （colony diam- eter Ф〈40 mm）, and there was no strain with fast growth rate （colony diameter Ф 〉 60 mm）. Pathogenicity test showed that the pathogenicity among strains was significantly different, only one strain had strong pathogenicity, and the others all had moderate or weak pathogenicity. [ Conclu- sion] The study confirmed the basic biological characteristics of the pathogen of rice sheath blight in Sichuan region, which would provide theoretical basis for effective control of rice sheath blight in the region.

Intron Retention Fine-Tunes the Resistance of the Rice Mutant pls4 to Rice Sheath Blight(Rhizotonia solani AG I.1a)

OsPLS4 encodes aβ-ketoacyl carrier protein reductase(KAR).The role of OsPLS4 in rice sheath blight(Rhizoctonia solani)remains unclear.Our preliminary studies showed that premature leaf senescence mutants(pls4)were highly susceptive to sheath blight in the early stage of rice development.To explore the role of this gene in the development of rice sheath blight,the transcriptome profiles of the rice pls4 mutant and wild type were compared by RNA-seq.The results revealed 2,569 differentially expressed genes(DEGs).The down-regulated genes were significantly enriched in the defense response-related biological processes.These down-regulated genes included the chitinase genes and WRKY genes,which were significantly changed in pls4 mutants.Furthermore,467 genes induced significant alternative splicing(AS)events.Among them,intron retention(IR)affected gene expression levels and functions of the vitamin B6(VB6)metabolism pathway related to sheath blight.This result suggests that IR plays an important role in the sheath blight resistance of mutant pls4.Together,these results indicate that pls4 could be involved in the biological process of sheath blight via DEGs and the fine-tuning of IR.The present study provides a molecular basis for further investigation of the resistance of rice to sheath blight.

Occurrence Regulation and Control Measure Comparison of Rice Sheath Blight

Rice sheath blight is widely distributed throughout the world, it can cause damage on many kinds of crops. Pathogen characteristics, infection cycle, occurrence law, damage law and different control methods of rice sheath blight in China are summarized, which will provide foundation for further worldwide research on the disease.

Efficacy of Difenoconazole-azoxystrobin 32.5% SC in Treatment of Rice Sheath Blight and Its Application Technology

[Objective] The efficacy of difenoconazole-azoxystrobin 32.5% SC for rice sheath blight and its application technology were discussed in this research. [Method] Three surveys were carried out. There were 5 fixed test clumps in each plot. The number of total plants and disease plants and disease progression in each fixed clump were recorded. The correlation effectiveness was calculated based on the growth rate of disease index. Significance analysis was performed with Dun- can＇s new multiple range method （DMRT）. [Result] The difenoconazole-azoxystrobin 32.5% SC had a good efficacy for rice sheath blight, and its efficacy increased with the increase of dose. If sprayed according to the dose of 450 ml/hm2 5 days before the beginning of heading stage of rice, difenoconazole-azoxystrobin 32.5% SC would have better effects on controlling rice sheath blight with correlation effectiveness reaching up to 95.14%, which was 2.06% higher than that of control drug （trifloxys- trobin-tebuconazole 75% WG, 225 g/hm2）. [Conclusion] Treated with difenoconazole- azoxystrobin 32.5% SC with dose of 450 ml/hm2, rice would have green upper leaves and less yellow middle and lower leaves. Moreover, the yield was in- creased significantly. The difenoconazole-azoxystrobin 32.5% SC had good safety. Therefore, difenoconazole-azoxystrobin 32.5% SC had a good application prospect in production.

Effect of Nitrogen Regimes on Grain Yield,Nitrogen Utilization,Radiation Use Efficiency,and Sheath Blight Disease Intensity in Super Hybrid Rice

Poor nitrogen use efficiency in rice production is a critical issue in China. Site-specific N managements （SSNM） such as real-time N management （RTNM） and fixed-time adjustable-dose N management （FTNM） improve fertilizer-N use efficiency of irrigated rice. This study was aimed to compare the different nitrogen （N） rates and application methods （FFP, SSNM, and RTNM methods） under with- and without-fungicide application conditions on grain yield, yield components, solar radiation use efficiency （RUE）, agronomic-nitrogen use efficiency （AEN）, and sheath blight disease intensity. Field experiments were carried out at Liuyang County, Hunan Province, China, during 2006 and 2007. A super hybrid rice Liangyou 293 （LY293） was used as experimental material. The results showed that RTNM and SSNM have great potential for improving agronomic-nitrogen use efficiency without sacrificing the grain yield. There were significant differences in light interception rate, sheath blight disease incidence （DI） and the disease index （ShBI）, and total dry matter among the different nitrogen management methods. The radiation use efficiency was increased in a certain level of applied N. But, the harvest index （HI） decreased with the increase in applied N. There is a quadratic curve relationship between grain yield and applied N rates. With the same N fertilizer rate, different fertilizer-N application methods affected the RUE and grain yield. The fungicide application not only improved the canopy light interception rate, RUE, grain filling, and harvest index, but also reduced the degree of sheath blight disease. The treatment of RTNM under the SPAD threshold value 40 obtained the highest yield. While the treatment of SSNM led to the highest nitrogen agronomic efficiency and higher rice yield, and decreased the infestation of sheath blight disease dramatically as well. Nitrogen application regimes and diseases control in rice caused obvious effects on light interception rate, RUE, and HI. Optimal N rate is helpful to get higher light interception rate, RUE, and HI. Disease control with fungicide application decreased and delayed the negative effects of the high N on rice yield formation. SSNM and RTNM under the proper SPAD threshold value obtained high-yield with high efficiency and could alleviate environmental pollution in rice production.

Efficacy of Bacillus subtilis MBI 600 Against Sheath Blight Caused by Rhizoctonia solani and on Growth and Yield of Rice

Rice sheath blight disease （ShB）, caused by Rhizoctonia solani, gives rise to significant grain yield losses. The present study evaluated the efficacy of Integral, the commercial liquid formulation of Bacillus subtilis strain MBI 600, against rice ShB and for plant growth promotion. In greenhouse studies, four log concentrations of Integral （from 2.2×10^6 to 2.2×10^9 cfu/mL） were used as seed treatment （ST）. After 25 d, seedlings were dipped （SD） into Integral prior to transplanting. At 30 d after transplanting （DAT）, leaf sheaths were inoculated with immature sclerotia of the pathogen. At 45 DAT, a foliar spray （FS） with Integral was applied to some treatments. The fungicide control was 50% carbendazim at 1.0 g/L, and a nontreated control was also included. Overall, there were 10 treatments, each with five replications. ShB severity was rated at 52 DAT, and seedling height and number of tillers per plant were rated at 60 DAT. In 2009, two field trials evaluated Integral at 2.2×10^8 and 2.2×10^9 cfu/mL. Integral was applied as ST, and seedlings were produced in a nursery bed. After 32 d, seedlings were treated with Integral as SD and transplanted into 10 m^2 blocks. Foliar sprays were given at 45 and 60 DAT. There were seven treatments, each with eight replications arranged as a factorial randomized complete block design. At 20 DAT, the plots were broadcast inoculated with R. solani produced on rice grains. Seedling height before transplanting, ShB severity at 90 DAT, and grain yield at harvest were recorded. Integral at 2.2×10^9 cfu/mL provided significant increase of seedling heights over other treatments under greenhouse conditions. The Integral treatments of ST ＋ SD ＋ FS at 2.2×10^9 cfu/mL significantly suppressed ShB over other treatments. In field studies, Integral provided significant increase of seedling height in nursery, and number of tillers per plant, compared with the control. ShB severity was significantly suppressed with higher concentrations of Integral compared to lower concentrations. Grain yield were the highest at an Integral concentration of 2.2×10^9 cfu/mL. Overall, Integral significantly reduced ShB severity, enhanced seedling growth, number of tillers per plant and grain yield as ST ＋ SD ＋ FS at the concentration of 2.2×10^9 cfu/mL under the conditions evaluated.

Analyses of the Temporal Development and Yield Losses due to Sheath Blight of Rice (Rhizoctonia solani AG1.1a)

Sheath blight of rice, caused by Rhizoctonia solani AG 1. 1a, has become the most important disease and caused serious yield losses in some major rice-growing regions in China in recent years. In the present study, field plot experiment was conducted to examine the relationships between disease intensity and inoculum density （ID）, the seasonal disease epidemic dynamics, and yield reductions due to disease damages. Results from the experiment demonstrated that the areas under progress curves of disease severity and those of percent rice tillers diseased were positively and closely related to the relative initial ID of the pathogen. The inoculum density-disease （IDD） relationships were simulated and the impractical linear models were obtained. Both logistic and Gompertz functions could be used to simulate the disease progress dynamics in time, but the progress curves of the disease severity were modeled better by the Gompertz than by logistic function. However, the Richards function was found to be the best in simulating the disease progress curves when a most appropriate value was chosen for the shape parameter m by using the computer software Epitimulator. Sheath blight infection decreased rice yield very significantly and a yield reduction of 40% was recorded in rice crop with the highest inoculum density. Rice yield was linearly and negatively correlated with the disease severity and the percent tillers affected. The simulated models for all these relationships were computed through executing Epitimulator software and were presented in this paper.

Secondary metabolites of rice sheath blight pathogen Rhizoctonia solani Kühn and their biological activities

Eight compounds were isolated from the fermentation cultures of rice sheath blight pathogen Rhizoctonia solani Kuhn. They were identified as ergosterol （1）, 6β-hydroxysitostenone （2）, sitostenone （3）, m-hydroxyphenylacetic acid （4）, methyl m-hydroxyphenylacetate （5）, m-hydroxymethylphenyl pentanoate （6）, （Z）-3-methylpent-2-en-1,5-dioic acid （7） and 3-methoxyfuran-2-carboxylic acid （8） by means of physicochemical and spectroscopic analysis. Among them, 2, 3, 5-8 were isolated from R. solani for the first time. All the compounds were evaluated for their biological activities. 4-6 and 8 showed their inhibitory activities on the radical and germ elongation of rice seeds. 1,4 and 7 showed moderate antibacterial activity to some bacteria. 4, 7 and 8 exhibited weak inhibitory activities on spore germination of Magnaporthe oryzae. 8 showed moderate antioxidant activity with the 1,1-diphenyl-2-picryhydrazyl （DPPH） andβ-carotene-linoleic acid assays. This is the first time to reveal compounds 5, 6 and 8 from rice sheath blight pathogen R. solani to have in vitro phytotoxic activity.

Gram-Positive Bacteria Associated with Rice in China and Their Antagonists Against the Pathogens of Sheath Blight and Bakanae Disease in Rice

It is necessary to understand the bacterial populations associated with rice so as to provide more information and natural resources for effective management of major diseases in rice. A survey on screening and identification of gram-positive bacteria was conducted during 1998 2004. Seven hundred and fifty-six rice samples were collected from Zhejiang, Jiangsu, Fujian and Yunnan Provinces, China. Over 1000 bacterial isolates were isolated and tested for colony morphology, pathogenicity, and some characteristics of bacteriology including Gram staining, fluorescent pigment on Kings medium B and microscopic observation for endospore. Together with five standard reference strains, 74 representative gram-positive bacterial isolates were confirmed by Biolog and gas chromatographic analysis of fatty acid methyl esters. Five bacterial species of Bacillus and other three genera were identified and isolates from Bacillus sublitis and Bacillus megaterium, exhibited the most effective inhibition against the pathogens of sheath blight and bakanae disease of rice. A few isolates from Bacillus pumilus and Bacillus megaterium showed weak virulent on rice together with some virulent isolates, risk should be considered when isolates from these species were screened for biocontrol agents.

Control Effects of Jinggangmycin and Its Compounded Reagents against Rice Sheath Blight

The control effects of six kinds of reagents against rice sheath blight were compared in the test. The results showed that 24% thifluzamide SC 300 mL/hm2 and 20% Jinggangmycin ~ prochloraz WP 750 g/hm2 had good control effect against rice sheath blight, and their control effects after spraying for 7 and 14 d were 72.16%, 77.91% and 66.52%, 71.08%, respectively. The control effects of both reagents were significantly better than other test reagents and control re- agent Jinggangmycin, and they had no adverse effect on growth and development of rice. Considering from the aspects of control effect and economy, 20% Jinggang- mycin ~ prochloraz WP was suggested to be used to control rice sheath blight, its recommended dosage was 600 -750 g/hm2, and the reagent volume was 750kg with coarse spraying.

Improvement of Rice Resistance to Sheath Blight by Pyramiding QTLs Conditioning Disease Resistance and Tiller Angle

Rice resistance to sheath blight （SB） is controlled by polygenes or quantitative trait loci （QTLs） and affected by plant morphological traits, qSB-9^TQ and TAG1^TQ, which control SB resistance and tiller angle （TA）, respectively, were employed to test whether the combination of the two genes will further improve rice SB resistance and reduce yield loss rather than only one of them or neither. Using two pairs of near isogenic lines （NILs）, TAC1^TQ was confirmed to contribute to SB resistance. However, its effect was less considerable than that of qSB-9^TQ. Subsequently, the two genes were introduced into two commercial rice varieties to develop a series of NILs. The NILs carrying both TAG1^TQ and qSB-9^TQ showed more resistance than the NILs containing only one of them. Comparing the grain yield per plant （GYP） under the three different SB disease conditions, namely slight, moderate and severe conditions, NILs carrying both genes apparently lost lower GYP than the NILs without the two genes and the NILs carrying only TAC1^TQ. Under slight disease condition, no significant differences were found on morphology, development and GYP associated traits except for TA between the NILs carrying both genes and either of them, indicating that the two genes have no inferior effect on rice agronomic traits. Results indicated that mAC1^TQ and qSB-9^TQ have high breeding potential, and pyramiding SB resistance QTL and morphological trait QTL is a potential approach in improving rice SB resistance.

Application Technology of Tebuconazole WG against Sheath Blight 75% Trifloxystrobin · Rice Blast and Rice

[ Objective] The paper was to confirm the best application period and frequency of 75% trifloxystrobin ·tebuconazole WG against rice blast and rice sheath blight. [ Method] Influences of different dosages and different application periods of 75% trifloxystrobin · tebuconazole WG on control effects against rice blast and rice sheath blight, as well as their effects on rice yields were studied in the paper. [Result] The control effects of three different fungicides application treatments against rice sheath blight were 80.24%, 83.0% and 67.99%, and the control effects against rice blast were 56.4%, 49.11% and 61.1%, respective- ly. Advanced application of fungicide for two times had good prevention effect against rice sheath blight ; properly delayed application of fungicides for two times was conducive to improving the control effect against rice blast, and one time application of sufficient fungicide during middle booting stage had higher control effect than application for two times. Effective panicle number per unit area, total grain number, 1 000-grain weight and moisture content of various fungicide application treat- ments did not have significant difference with those of the treatments without fungicide application, but grain number per panicle in treatments applied with fungicide was higher than the treatment without application. [ Conclusion] 75% Trifloxystrobin ·tebuconazole WG has better control effect on rice blast and sheath blight, which helps to promote the formation of rice grain and increases yield significantly. The fungicide application against rice sheath blight should be appropriately ad- vanced, and application for one time against panicle blast after middle booting stage is helpful to improve the control effect.

Effect of Morphological Traits on Sheath Blight Resistance in Rice

Sheath blight, caused by the Rhizoctonia solani Kuhn, is one of the most serious diseases of rice (Oryza sativa L.). The relationship between morphological traits and sheath blight resistance was investigated in 1999 and 2000 by using a segregant population consisting of 240 inbred recombination lines, derived from an elite combination of Zhenshan 97 X Minghui 63. Partial correlation analysis between phenotypic data (morphological characteristics and heading date (HD)) and sheath blight ratings (SBR) showed that only one trait, plant compactness, was significantly correlated with resistance to sheath blight in both 1999 and 2000, eight traits, such as plant height, heading date, and penultimate leaf angle, were significantly correlated with SBR in either of the two years, and the remaining 10 morphological traits were not consistently associated with SBR in the two years. A major quantitative trait locus (QTL) controlling plant compactness was found to be in the site on chromosome 9 adjacent to the region responsible for qSB9, a major QTLs conferring sheath blight resistance. One out of the three QTLs contributing to a penultimate leaf angle was mapped approximately on the same region as another sheath blight resistance QTL, qSB5, located on chromosome 5. Whereas, no QTLs underlying most other traits was detected on the chromosomal region correlated with sheath blight resistance QTL. The results in the present study suggested that the morphological traits were not the main factors responsible for the SBR separation among the recombinant inbred lines (RILs) population. Nevertheless, some morphological characteristics had some indirect influence on expression of sheath blight resistance genes through altering a microclimate on paddy field so as to influence the infection of the pathogen and development of the disease. An efficient approach in resistance breeding to sheath blight was recommended by pyramiding major QTLs for sheath blight resistance and selecting those morphological traits that favor resistance gene expression.

The Effect of Three New Fungicides against Rice Sheath Blight in Field Experiment

[Objective] The aim was to study the effect of three new fungicides against rice sheath blight in field experiment. [Methods] The experiment set up 7 treatments with three times of repetition and designed by random grouping. By using 5 sampling points in each plot, and investigating continuous 4 holes of each point, total plants, diseased plants and disease degrees were recorded. Then disease index and control efficiency were calculated, and variance analysis was carried out. [Results] 300 or 450 ml/hm^2 azoxystrobin ＋ difenoconazole 325 g/L SC had better control efficiency to rice sheath blight and had no phytotoxicity effect, we should use it at the initial disease stage and continuously spray 2-3 times. [Conclusion] The experiment provided a theoretical basis for controlling rice sheath blight using fungicides.

Mapping QTLs Associated with Sheath Blight Resistance Using Chromosome Segment Substitution Lines of Rice(Oryza sativa L.)

In this study, a population of 119 chromosome segment substitution lines （CSSLs） derived from backcross between indica 9311 and japonica Nipponbare was employed to map quantitative trait loci （QTL） associated with sheath blight resis-tance in rice with toothpick inoculation method. A total of three sheath blight resis-tance-associated QTLs （qsb8-1, qsb8-2 and qsb8-3） were identified, which were lo-cated on adjacent molecular markers RM3262, RM5485 and RM3496 of chromo-some 8; the genetic interval was 81.7cM-91.7cM, 91.7cM-108.1cM and 108.1cM-119.6cM, respectively. The additive effect of qsb8-2 was negative, indicating that sheath blight resistance of susceptible parent harboring qsb8-2 fragment was en-hanced; additive effects of qsb8-1 and qsb8-3 were positive, indicating that sheath blight resistance of susceptible parent harboring qsb8-1 and qsb8-3 fragments was reduced.

Control Efficacy of Validamycin A·(0.2 Billion Spores/ml) Paenibacillus polymyxa DN-1 3% AS for Rice Sheath Blight

With validamycin A.（0.2 billion spores/ml） Paenibacillus polymyxa DN-1 3% AS as the test agent, the effects of different dosage and different application time on the control efficacy for＇ rich sheath blight were investigated. The results of two- year test showed that when the application amount was in the range of 45-90 g.a.i/ hm^2, the field efficacy of validamycin A-（0.2 billion spores/ml） P. polymyxa DN-1 3% AS in the initial infection stage of rich sheath blight （Le., the diseased plant rate was below 5%） reached 80.38%-89.06%, and that in the peak infection stage （i.e., the diseased plant rate was higher than 10%） reached only 41.12%-53.26%. The field efficacy of validamycin A.（0.2 billion spores/ml） P. polymyxa DN-1 3% AS at the early onset of rich sheath blight was significantly better than that at the onset, so that the application time of validamycin A .（0.2 billion spores/ml） P. polymyxa DN-1 3% AS should be appropriately brought forward in the prevention and control of rice sheath blight.

Prospect of the QTL-qSB-9^(Tq) utilized in molecular breeding program of japonica rice against sheath blight

The major QTL-qSB-9^Tq conferring partial resistance to rice （Oryza sativa L.） sheath blight （Rhizoctonia solani Kvhn） has been verified on chromosome 9 of the indica rice cultivar, Teqing. In this study, the prospect of this QTL utilized in molecular breeding program of japonica rice for sheath blight resistance was investigated. Most of the japonica rice cultivars showed lower level of sheath blight resistance than the indica rice cultivars. At the corresponding site of qSB-9^Tq, nine typical japonica rice cultivars from different ecological regions or countries proved to possess the susceptible allele（s）. Introgression of qSB-9^Tq into these cultivars enhanced their resistance level by decreasing sheath blight score of 1.0 （0.5-1.3）, which indicated that qSB-9^Tq had a large potential in strengthening the resistance of japonica rice to sheath blight. The use of the three molecular markers, which were polymorphic between Teqing and many japonica rice cultivars, promotes the application of qSB-9^Tq in a concrete molecular breeding program.

Identification of New Resistance Loci Against Sheath Blight Disease in Rice Through Genome-Wide Association Study

Sheath blight(SB) caused by the soil borne pathogen Rhizoctonia solani is one of the most serious global rice diseases. Breeding resistant cultivar is the most economical and effective strategy to control the disease. However, no rice varieties are completely resistant to SB, and only a few reliable quantitative trait loci(QTLs) linked with SB resistance have been identified to date. In this study, we conducted a genome-wide association study(GWAS) of SB resistance using 299 varieties from the rice diversity panel 1(RDP1) that were genotyped using 44 000 high-density single nucleotide polymorphism(SNP) markers. Through artificial inoculation, we found that only 36.5% of the tested varieties displayed resistance or moderate resistance to SB. In particular, the aromatic and aus sub-populations displayed higher SB resistance than the tropical japonica(TRJ), indica and temperate japonica sub-populations. Seven varieties showed similar resistance levels to the resistant control YSBR1. GWAS identified at least 11 SNP loci significantly associated with SB resistance in the three independent trials, leading to the identification of two reliable QTLs, qSB-3 and qSB-6, on chromosomes 3 and 6. Using favorable alleles or haplotypes of significantly associated SNP loci, we estimated that both QTLs had obvious effects on reducing SB disease severity and can be used for enhancing SB resistance, especially in improving SB resistance of TRJ sub-population rice varieties. These results provided important information and genetic materials for developing SB resistant varieties through breeding.

Advances in Mapping Loci Conferring Resistance to Rice Sheath Blight and Mining Rhizoctonia solani Resistant Resources

Sheath blight （SB） caused by Rhizoctonia solani is one of the three major diseases of rice, and now has become the most severe disease causing rice yield loss in China. Breeding and use of varieties resistant to SB is crucial in controlling the disease, but the advances achieved have been limited due to the tack of highly SB-resistant rice germplasm. Genetic analysis revealed that the SB resistance in rice was a typical quantitative trait controlled by multi-genes. Although many QTLs conferring resistance to SB have been identified in recent years, most of the QTLs only showed small effects and few of them have been evaluated for utilization potential. Many R. solani-resistant resources have been found in wild rice species, microorganisms and other plant species. It is already known that the SB-resistance could be improved in transgenic rice plants by genetic transformation. This paper reviewed the genetic mapping of loci associated with resistance to rice SB, the evaluation of the potential of resistance QTLs, and the resistant resources found in various organisms besides rice. To develop SB-resistant rice varieties, it is important to develop and explore new resistant rice germplasms, fine map and evaluate resistance QTLs, and also to pay attention to various bio-resources showing resistance to R. solani.

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.