Pathogenesis-related gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae

Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between Pathogenesis Related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, and OsPR1c genes were cloned and their induction was analyzed, in addition to the OsPR10a gene, at the juvenile and adult stages in response to a wildtype Xoo strain that induces a resistance response (incompatible interaction) and an isogenic mutant Xoo strain that does not (compatible interaction). We found that the adult stage leaves are more competent to express these OsPR1 genes and that the Xa21 locus is required for the highest levels of induction.     

Pathogenesis-related gene expression in rice is correlated with developmentally controlled Xa21-mediated resistance against Xanthomonas oryzae pv. oryzae

Disease resistance mediated by the resistance gene Xa21 is developmentally controlled in rice. We examined the relationship between Pathogenesis Related (PR) defense gene expression and Xa21-mediated developmental disease resistance induced by Xanthomonas oryzae pv. oryzae (Xoo). OsPR1a, OsPR1b, and OsPR1c genes were cloned and their induction was analyzed, in addition to the OsPR10a gene, at the juvenile and adult stages in response to a wildtype Xoo strain that induces a resistance response (incompatible interaction) and an isogenic mutant Xoo strain that does not (compatible interaction). We found that the adult stage leaves are more competent to express these OsPR1 genes and that the Xa21 locus is required for the highest levels of induction.     

Characterization and host range of five tumorigenic Agrobacterium tumefaciens strains and possible application in plant transient transformation assays

Agrobacterium tumefaciens is a Gram‐negative bacterium. It causes plants to produce crown gall disease because of the transfer, integration and expression of oncogenes encoded by the T‐DNA (transferred DNA) region of the tumour‐inducing (Ti) plasmid. A set of transferred genes directs the production of bacterial nutrients, called opines, formed by condensation of an amino acid and a keto acid or a sugar. Transformed cells synthesize and secrete substantial quantities of particular opines, which A. tumefaciens then uses as a carbon and sometimes nitrogen source. Agrobacterium tumefaciens strains are usually classified on the basis of the opines they can catabolize. Because of the ability to transfer DNA between different kingdoms, A. tumefaciens is also frequently used to generate transgenic plants. This study analyses five poorly characterized wildtype Agrobacterium strains, 1D1108, 1D1460, 1D132, 1D1478 and 1D1487, isolated from Euonymus, cane, cherry, Salix and apple, respectively. Partial Ti‐plasmid sequence analysis demonstrated that the five strains harbour the nopaline‐type Ti plasmid. Tumorigenesis and transient transformation assays of the five analysed and six wildtype A. tumefaciens strains were performed with selected plant species, including two or three species of Brassicaceae, Asteraceae, Solanaceae, Apiaceae and Leguminosae. The A. tumefaciens strains 1D1108, 1D1460 and 1D1478 showed higher transformation efficiencies than the previously characterized A. tumefaciens strains with several economically important crops. These data suggest the potential use of these newly characterized wildtype A. tumefaciens strains in transient transformation assays with certain plant species.     

High activities and mRNA expression of pyrophosphate-fructose-6-phosphate-phosphotransferase and 6-phosphofructokinase are induced as a response to Rhizoctonia solani infection in rice leaf sheaths

Rice sheath blight disease caused by Rhizoctonia solani Kuhn results in significant yield and quality losses in rice growing areas worldwide. The glycolytic pathway is important in the resistance response to R. solani infection in rice. This study examined one of the regulatory steps in this pathway catalyzed by pyrophosphate-fructose-6-phosphate-phosphotransferase (PFP) and 6-phosphofructokinase (PFK). PFP and PFK activity in R. solani-infected rice plants increased. The mRNA expression of PFP/PFK isozymes showed that PFK 1, 2, 4 and 5 in the resistant line at 1 dpi were high as compared to the gradual increase observed in the expression of all PFP isozymes. Also PFK 1, PFK 3, PFK 4, PFK 5, PFP 2 and PFP 5 were adaptive to sheath blight disease infection and linked to defence response while, the expressions of PFK 2, PFP 1, PFP 3 and PFP 4 although adaptive, were not specific to R. solani infection. These observations provide evidence that (a) both PFP and PFK have isozymes that play an adaptive role after R. solani infection but while those of PFK are expressed at higher levels within a short time after infection those of PFP are expressed gradually, (b) the adaptive activation of PFP in R. solani-infected rice plants is correlated with the paired expression of its α- and β-subunits as shown by PFP 2 and PFP 5, and (c) the expression of some α-subunits is not specific to R. solani infection as shown by PFP 1, PFP 3 and PFP 4.     

表达Harpin蛋白质Hpa1功能片段的转基因小麦对赤霉病的抗性

来自水稻黄单胞菌的harpin蛋白质Hpa1有促进植物生长、诱导植物抗病性的功能,Hpa1序列10~42氨基酸片段（Hpa1_10-42）的活性比全长分子高1.3~7.5倍。为研究Hpa1_10-42在转基因小麦体内表达以后对赤霉病的影响和评价转基因小麦抗病水平与应用潜力,对6个小麦转基因系进行了测定。结果显示,Hpa1_10-42在转基因系T₃~T₅代呈现稳定表达,用禾谷镰刀菌一个分离物进行接种以后,转基因系发生赤霉病的程度较非转基因小麦显著降低,且转基因系T₃~T₅代赤霉病降低的趋势一致。另外,转基因系小麦病害减轻的程度,与在非转基因小麦上使用杀菌剂的效果相当,表明Hpa1_10-42转基因表达对小麦赤霉病有防治作用。     

非亲和性稻瘟病菌对水稻的诱导抗性

为研究稻瘟病菌Magnaporthe oryzae不同菌株间的相互作用,选择与单抗性基因系水稻IRBL5-M （携带抗性基因Pi5）表现为亲和性的菌株HN52与非亲和性的菌株HN119为研究对象,将其单独或混合接种到单抗性基因系水稻IRBL5-M中,并通过荧光显微镜观察接种后水稻叶鞘的发病情况及病斑面积,测定接种后水稻内相关抗性基因OsWRKY45、OsNPR1、OsPR10、OsMAPK2的表达量以及活性氧的变化。结果显示,相较于单独接种亲和性菌株,混合接种后单抗性基因系水稻IRBL5-M病斑发病面积减少;混合接种中亲和性菌株HN52菌丝侵染能力降低,侵染菌丝细胞间扩展率显著降低73.13%;同时单抗性基因系水稻IRBL5-M中OsWRKY45、OsNPR1、OsPR10和OsMAPK2抗性基因表达量显著增加,水稻叶片中活性氧含量增加,表明在菌株混合侵染过程中,非亲和性菌株可通过激发水稻的抗性反应来降低亲和性菌株对水稻的侵染程度。     

哈尔滨地区抗瘟基因抗性分析及水稻品种抗性评价与利用

为明确哈尔滨地区24个抗瘟基因的抗性和20个水稻品种含有抗瘟基因型的情况,选取200个稻瘟病菌单孢菌株和22个已知无毒基因的鉴别菌株作为鉴定系统,通过喷雾法和离体划伤法接种,应用PCR技术对部分试验结果进行了重复性检测,并在此基础上进行了抗性布局分析。结果表明:24个抗瘟基因抗性差异较大,抗谱在11.00%~93.00%之间,平均抗谱38.29%;抗谱超过80.00%的基因有3个,由高到低为Pi-9、Pi-ta2和Pi-z5;抗谱低于20.00%的基因有9个,分别为Pi-a、Pi-ks、Pi-i、Pi-t、Pi-kp、Pi-ta、Pi-3、Pi-km和Pi-sh,Pi-a抗性最差。20个水稻品种中检测到抗瘟基因13个,共出现40次,其中Pi-a和Pi-19各出现6次,频率最高;龙稻5含抗瘟基因最多,4个,分别为Pi-ks、Pi-1、Pi-19和Pi-sh。在哈尔滨晚熟稻区建议布局绥粳8、中龙香粳1或者绥粳8+中龙香粳1搭配;在早熟稻区建议布局龙粳18、哈香07-321或者龙粳18+哈香07-321搭配。     

转cry1Ab基因粳稻对稻纵卷叶螟成虫产卵行为的影响

为评价转cry1Ab基因粳稻(KMD1和KMD2)对稻纵卷叶螟Cnaphalocrocis medinalis(Guenée)成虫产卵行为的影响,采用"Y"型嗅觉仪和笼罩以及田间试验等方法对稻纵卷叶螟成虫对转基因水稻的趋性以及其产卵选择性进行了研究,并利用固相微萃取和GC-MS技术测定了这2种Bt水稻及其对照亲本秀水11挥发物的组成及其相对含量。结果表明,稻纵卷叶螟成虫在"Y"型嗅觉仪和小型笼罩内对Bt水稻和对照亲本的选择性差异不显著,而在大型笼罩和田间条件下均显著趋向于在非转基因水稻上产卵,其中大田中稻纵卷叶螟在KMD1、KMD2和对照中的百叶卵量分别为2.20±1.28、1.00±0.77和5.60±2.16粒。水稻挥发物的组成及其相对含量在Bt水稻及其对照亲本之间差异不显著。表明相对于Bt水稻,在田间条件下稻纵卷叶螟成虫趋向于在非转基因水稻产卵,而水稻挥发物可能不是引起这种行为趋性的直接原因。     

Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice

Protoporphyrinogen oxidase (Protox) of Myxococcus xanthus (Mx Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, Mx Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the Mx Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the Arabidopsis Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified Mx Protox gene was used to generate transgenic rice plants via Agrobacterium-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content Mx Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native Mx Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.     

水稻纹枯病菌拮抗细菌的筛选及鉴定

为获得对水稻纹枯病有生防效果的拮抗细菌,从江苏南京、徐州和常州等地采集的土样中分离细菌分离物1914株,采用平板对峙法筛选获得70株对水稻纹枯病菌有较强抑菌活性的分离物,其中11株对5种水稻病害病原菌均有抑制作用;对11株拮抗菌进行田间防效和室内促生试验,测定菌株分泌的抑菌物质和促生物质,并进行种属鉴定.结果表明,拮抗菌对水稻纹枯病的盆栽和田间小区防效在48.41％和43.03％以上;均可产生蛋白酶与嗜铁素,而不产生几丁质酶,除XF-174外其余10个菌株均可产生纤维素酶;对水稻苗株高和鲜重具有促生作用,并均可产生赤霉素(GA3);除ZF-273和XF-174外的9个菌株可产生吲哚乙酸(IAA),且细菌发酵液中IAA和GA3含量与水稻株高和鲜重的增长率呈正相关.结合各菌株形态特征、生理生化特性和16S rDNA与gyr-B序列分析结果,鉴定SF-181为枯草芽胞杆菌Bacillus subtilis,XF-174为荧光假单胞菌Pseudomonas fluorescens,其余9个菌株为解淀粉芽胞杆菌B.amyloliquefaciens.     

Effect of temperature on toxicity of pyrethroids and endosulfan, activity of mitochondrial Na-K-ATPase and Ca-Mg-ATPase in Chilo suppressalis (Walker) (Lepidoptera: Pyralidae)

The toxicity of deltamethrin, bifenthrin, and endosulfan to the fourth-instar larvae of rice stem borer, Chilo suppressalis (Walker), was measured at 17, 27, and 37 °C. The three insecticides all displayed a positive temperature coefficient between 17 and 37 °C. The temperature coefficients of deltamethrin, bifenthrin, and endosulfan were 5.59, 1.68, and 2.85, respectively. However, temperature coefficients of deltamethrin and bifenthrin between 17 and 27 °C and between 27 and 37 °C varied. The inhibition of the above three insecticides to mitochondrial Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase from the fourth-instar larvae of rice stem borer was determined at 17, 27, and 37 °C. The inhibition of deltamethrin to the specific activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase showed a negative temperature coefficient, but endosulfan exhibited a positive temperature coefficient. Inhibition of bifenthrin exhibited the contrary temperature coefficients between Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase and a negative temperature coefficient for the former and a positive temperature coefficient for the later.     

菜豆普通细菌性疫病菌在土壤和植株残体中的越冬能力

为评估菜豆普通细菌性疫病菌地毯草黄单胞杆菌菜豆致病变种或褐色黄单胞菌褐色亚种在土壤及植物残体中的越冬能力,对采自黑龙江、内蒙古、山西、河北及新疆的18块菜豆生产田的20份土壤及14份植物残体样品进行病原菌分离和鉴定。在MT选择性培养基上有12个土壤样品和13个植株残体样品提取液产生典型的类似黄单胞菌菌落。选取29个分离物进行致病性测定,有27个分离物对菜豆品种"英国红"致病。利用地毯草黄单胞杆菌菜豆致病变种和褐色黄单胞菌褐色亚种的特异性引物X4c/X4e及褐色黄单胞菌褐色亚种特异性引物Xf1/Xf2对29个分离物进行多重PCR检测,其中17个分离物为地毯草黄单胞杆菌菜豆致病变种,10个分离物为褐色黄单胞菌褐色亚种。结果表明,菜豆普通细菌性疫病菌可以在黑龙江、内蒙古、山西、河北的一些菜豆种植区的土壤及植株残体中越冬存活。     

Effects of different treatment methods of the fungicide jinggangmycin on reproduction and vitellogenin gene (Nlvg) expression in the brown planthopper Nilaparvata lugens Stål (Hemiptera:Delphacidae)

Jinggangmycin is an antibiotic fungicide against the rice sheath blight, Rhizoctonia solani, in China. Previous investigations have shown that jinggangmycin leaf spray stimulated the fecundity of Nilaparvata lugens Stål (Hemiptera:Delphacidae), but the effects of different application methods and concentrations of jinggangmycin on the reproduction of N. lugens have not been investigated. Here, we investigated three treatment methods (foliar and stem sprays and topical treatment) and four concentrations (100, 200, 400 and 800 ppm) of jinggangmycin on the reproduction of adult female N. lugens. The results showed that leaf spray significantly stimulated the fecundity of N. lugens and increased the vitellin content in female ovaries and the gene expression level of vitellogenin (Nlvg), but there was no significant effect on reproduction found for stem spray. In four concentrations of jinggangmycin, leaf sprays at concentrations of 100, 200 and 400 ppm and topical treatments of 100 and 200 ppm significantly increased the number of eggs laid, the vitellin content and the gene expression level of Nlvg compared to the control. Therefore, we suggest that stem spray at high concentration should be applied (if possible) when jinggangmycin is used against rice sheath blight to facilitate harmonious control of rice pests.     

Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

Characterization of glutathione S-transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS-polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3-10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.     

Influence of rice development on the function of bacterial blight resistance genes

Disease resistance genes most commonly used in breeding programs are single, dominant genes with relative effectiveness that is sometimes influenced by plant developmental stage. Knowing the developmental stages at which a resistance gene is functional is important for disease management. In rice, resistance at the seedling stage is crucial, because wounding during transplanting increases the potential for bacterial blight disease, and not all bacterial blight resistance genes are effective at the seedling stage. Effectiveness of the bacterial blight resistance genes Xa4, xa5, and Xa7, all in a common genetic background, was evaluated at different developmental stages by measuring lesion length and bacterial numbers after inoculation with the bacterial pathogen, Xanthomonas oryzae pv. oryzae. The Xa4 and xa5 genes controlled disease at all growth stages. In contrast, Xa7 was not fully functional in very young seedlings, but was completely effective by 21 days after sowing (das). The effects of plant developmental stage on interactions of the Xa7 gene with X. oryzae pv. oryzae strains carrying different mutant avrXa7 alleles were also tested. If a partial or fully functional avrXa7 allele was present, Xa7 resistance was effective at all growth stages tested after the transplant stage (>21 das).     

Two cytochrome P450 genes are involved in imidacloprid resistance in field populations of the whitefly, Bemisia tabaci, in China

The sweet potato whitefly, Bemisia tabaci (Gennadius) (Hemiptera:Aleyrodidae), is an invasive and damaging pest of field crops worldwide. The neonicotinoid insecticide imidacloprid has been widely used to control this pest. We assessed the species composition (B vs. Q), imidacloprid resistance, and association between imidacloprid resistance and the expression of five P450 genes for 14–17 B. tabaci populations in 12 provinces in China. Fifteen of 17 populations contained only B. tabaci Q, and two populations contained both B and Q. Seven of 17 populations exhibited moderate to high resistance to imidacloprid, and eight populations exhibited low resistance to imidacloprid, compared with the most susceptible field WHHB population. In a study of 14 of the populations, resistance level was correlated with the expression of the P450 genes CYP6CM1 and CYP4C64 but not with the expression of CYP6CX1, CYP6CX4, or CYP6DZ7. This study indicates that B. tabaci Q has a wider distribution in China than previously reported. Resistance to imidacloprid in field populations of B. tabaci is associated with the increased expression of two cytochrome P450 genes (CYP6CM1 and CYP4C64).     

Pi34-AVRPi34: a new gene-for-gene interaction for partial resistance in rice to blast caused by Magnaporthe grisea

The japonica rice (Oryza sativa) cultivar Chubu 32 has a high level of partial resistance to blast, which is mainly controlled by a dominant resistance gene located on chromosome 11. The partial resistance to the rice blast fungus (Magnaporthe grisea) in Chubu 32 has isolate specificity; isolate IBOS8-1-1 is more aggressive on Chubu 32 than are other isolates. We hypothesized that the gene-for-gene relationship fits this case of a partial resistance gene in Chubu 32 against the avirulence gene in the pathogen. The partial resistance gene in Chubu 32 was mapped between DNA markers C1172 (and three other co-segregated markers) and E2021 and was designated Pi34. In the 32 F₃ lines from the cross between a chromosome segment substitution line (Pi34⁻) from Koshihikari/Kasalath and Chubu 32, the lines with high levels of partial resistance to the M. grisea isolate Y93-245c-2 corresponded to the presence of Pi34 estimated by graphic genotyping. This indicated that Pi34 has partial resistance to isolate Y93-245c-2 in compatible interactions. The 69 blast isolates from the F₁ progeny produced by the cross between Y93-245c-2 and IBOS8-1-1 were tested for aggressiveness on Chubu 32 and rice cultivar Koshihikari (Pi34⁻). The progeny segregated at a 1 : 1 ratio for strong to weak aggressiveness on Chubu 32. The results suggested that Y93-245c-2 has one gene encoding avirulence to Pi34 (AVRPi34), and IBOS8-1-1 is extremely aggressive on Chubu 32 because of the absence of AVRPi34. This is the first report of a gene-for-gene relationship between a fungal disease resistance gene associated with severity of disease and pathogen aggressiveness.     

稻飞虱及黑肩绿盲蝽在探照灯下的扑灯节律

为明确稻飞虱及其天敌黑肩绿盲蝽在探照灯下的扑灯节律,2012和2013年在广西兴安县应用探照灯诱虫器对稻飞虱及黑肩绿盲蝽进行了分时段监测。结果表明：稻飞虱和黑肩绿盲蝽年度间扑灯量有所不同,夜晚扑灯类型属于整夜扑灯型,晚上一般有1~2个扑灯高峰。2012年稻飞虱和黑肩绿盲蝽、2013年白背飞虱在迁入高峰期扑灯高峰不固定,2013年褐飞虱扑灯高峰主要集中在19：30～21：00时段,黑肩绿盲蝽2013年扑灯高峰主要集中在00：00～01：30和01：30～03：00时段。稻飞虱和黑肩绿盲蝽迁出高峰期及黑肩绿盲蝽本地活动期扑灯高峰主要集中在前半夜,稻飞虱本地活动期扑灯高峰主要集中在前半夜和04：30～06：00时段。     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.