Fusarium head blight caused by Fusarium spp. occurs worldwide on small grain cereals. In China, Fusarium head blight is mainly caused by Fusarium graminearum species complex(FGSC). FGSC produces trichothecene toxins and it was divided into 3 chemotypes: nivalenol(NIV), 15-acetyldeoxynivalenol(15-AcDON) and 3-acetyldeoxynivalenol(3-AcDON). In order to distinguish these three chemotypes, Tri8 gene of three chemotype isolates was amplified by using three pairs of primers and sequenced. According to the difference of Tri8 gene sequences of three chemotypes, specific primers were designed and screened. We found that 3 chemotypes can be differentiated according to the size of fragments after Tri8 gene was digested with AvaI enzyme.The procedure developed in this study could be used to identify the chemotype of the major pathogen of Fusarium head blight in China.

In this study, isolation and identification of the pathogens causing maize ear rot were conducted from the samples collected from spring maize regions in 2015.The results demonstrated that Fusarium graminearum species complex in which most of them from Shanxi, Hebei, Jilin and Heilongjiang accounting for 81.25%, 75.00%, 44.00% and 44.44%, respectively, predominated in this region with the total isolation frequency of 35.90%.In addition, F. verticillioides was the dominant species in Inner Mongolia with arate of 56.25%.In Liao-ning Province, F. verticillioides and Trichoderma harzianum were the main pathogens with isolation frequencies of 34.48% and 31.03%, respectively. And, in Shaanxi Province, F. graminearum species complex, F. verticillioides and F. subglutinans showed up with the same frequency of 28.57%.For subspecies identification of the F. graminearum species complex, a phylogenetic analysis based on the EF-1α gene sequences revealed that F. graminearum and F. boothii were present in spring maize region with F. boothii as the dominant species.

The pink mold rot on traditional fruit tree Ziziphus jujube was observed in plantation of Shanxi pro-vince. At the beginning, pink mold rot appeared as white mold on the fruit surface. As the disease progressed, it changed to thick and larger powdery mildew lesion with variation in the size. The appearances were punctiform, mottling, patching, ellipse and so on. The morphological characteristics, pathogenicity test and molecular identification method were used to confirm the pathogen of pink mold rot. Two representative strains of Trichothecium roseum were identified in this study based on morphological and physiological tests in addition to the ITS sequence comparison. The conidia of T. roseum germinated with optimal conditions at 20℃-25℃, pH 4-10, and over 75% relative humidity. The lethal temperature for conidia of T. roseum was 48℃ for 10 minutes. The germ tube of T. roseum grew with an optimal condition at 20℃-25℃, pH 6, more than 75% relative humidity. The results of this research provide the theoretical basis for disease diagnosis and control measures.

Bacterial stem and root rot of sweet potato is a severe disease which can infect its stems, petioles, leaves and tuberous roots. The typical symptoms of the disease include black and rot basal stems, yellow leaves, dark brown vascular stems and smelly tuberous roots. On the basis of typical symptoms, pathogen isolation, purification and pathogenicity test, it was confirmed that it was a bacterial disease. Through the observation of the bacterial morphological and cultural characteristics, it was found that the pathogen is a short rod-shaped, gram-negative bacterium with peritrichous flagella, 2.36 μm × 0.4 μm in size, and can elicit the hypersensitive response (HR) in tobacco. According to Biolog test, analysis of fatty acid methyl esters and 16S rDNA sequence, MALDI-TOF mass spectrometry test and phylogenetic sequence analysis of 8 house-keeping genes, dnaX, rplB, fusA, gapA, gyrA, purA, recA and rpoS, it was found that the pathogen shares high similarity with Dickeya dadantii. These results indicated that the stem rot disease of sweet potato in Zhejiang Province was caused by D. dadantii.

To identify the viruses that may cause Lycopersicon esculentum malformations wrinkling disease,we used biological, serological and molecular methods. Results revealed that the virus associated with this disease was Cucumber mosaic virus (CMV). Two isolates were identified as SXFQ (GenBank accession number: JX993914) and FQ (GenBank accession number: JX993912). The identities of nucleotide and deduced amino acid sequences of CP gene among 9 CMV isolates of our research group and 3 other typical CMV isolates ranged from 77.1 % to 100 % and 81.6 % to 100 %, respectively. Phylogenetic trees based on the aa sequences showed that 9 CMV isolates of our laboratory belong to two branches of CMV subgroup Ⅰ B. SXFQ and other 5 isolates belong to a branch which exhibits strong symptoms on the indicator plant. FQ and other 2 isolates belong to another branch which exhibits weak symptoms on the indicator plant. We analysed the subgroup of 9 Shanxi isolates CP. The results showed that the physical and chemical properties, stability and hydrophobicity were similar. The two branches showed similar amino acid variation and protein structure. It indicates that CMV isolates of the same branch of CMV subgroup Ⅰ B had some nearly certain regularity.

One hundred and thirty-five Phyllosticta isolates collected from main citrus producing regions across China, and Africa, Australian, America were amplified using 11 ISSR primers. PCR products were electrophoresed at 95V in a 1.5% agarose gel for 90 min, and then stained with ethidium bromide. The clustering analysis was performed using NTSYSpc version 2.10 software. The amplification results showed that 116 fragments were amplified, and 112 fragments displayed polymorphism that accounted for 96.55% in the total amplified fragments. The average number of bands amplified per primer was 10.55 and the length of the amplified fragments ranged from 250 to 3 000 bp. UPGMA cluster analysis based on ISSR data showed that Phyllosticta isolates collected from the citrus main producing regions in China were divided into four groups, that were associated with species of P. citricarpa, P. citriasiana, P. capitalensis and P. citrichinaensis, respectively. The group of P. citricarpa was divided into 5 subclades at a level of 0.97 similarity coefficient. Subclade I consisted of 28 isolates was collected from Citrus reticulata cv. Bendizao, Citrus unshiu, C. reticulata cv. Manju and C. reticulata cv. Nanfengmiju. Subclade II consisted of 5 isolates was collected from C. reticulata cv. Shatangju. Subclade III consisted of 7 isolates was collected from Mozambique lemon and grapefruit, American orange, South Africa orange and Australian citrus. Subclade IV consisted of 13 isolates was collected from sweet orange in China. Subclade V consisted of 9 isolates was collected from lemon in China. These results showed that isolates of P. citricarpa collected from China had abundant genetic variation, and the genetic variation of P. citricarpa was relevant to its hosts. It may be different in origin between the pathogen of citrus black spot in China and the pathogen of citrus black spot in foreign countries.There was genetic variation within populations of P. citriasiana, P. capitalensis and P. citrichinaensis, but the genetic variation might be not relevant to its geographic distribution or hosts. The results would lay the foundation for the study of the genetic diversity of Phyllosticta species associated with citrus.

In this study, we developed a rapid, accurate, and sensitive assay for detection of F. sambucinum based on loop-mediated isothermal amplification (LAMP) technology. By comparing different candidate gene sequences among F. sambucinum and its relative species, TEF1-α (translation elongation factor 1-α) was selected as a target to design the LAMP primers for specific detection of F. sambucinum. The developed LAMP assay can efficiently amplify the target gene in 70 min at 62℃. By adding HNB (hydroxynaphthol blue) prior to the assay, the result can be directly visualized through eyes. A positive reaction exhibited sky blue color, while purple color corresponds to a negative reaction. The results showed specificity for F. sambucinum rather than the other assayed fungal isolates. The lowest detectable limit of the LAMP assay for F. sambucinum DNA was 100 pg·μL^-1. Among 28 suspected diseased samples of potato dry rot collected from Inner Mongolia and Heilongjiang provinces, F. sambucinum was detected in 14 samples. This technology can be used for fast and accurate detection of F. sambucinum and diagnosis of F. sambucinum-caused potato dry rot.

OsNAC3 is a member of NAC transcription factor family in rice and induced by MeJA, drought, salt or cold treatments. OsNAC3 and GFP fusion protein was targeted to the nucleus. The OsNAC3 protein showed transactivation activity at its C-terminus in yeast and formed homocomplexes by yeast two-hybrid assay and pull down assay.Electrophoretic mobility shift assays demonstratedthat the recombinant OsNAC3 protein could bind specifically to NAC recognized sequence (NACRS) in vitro. The results provide some data for further understanding the biological function of OsNAC3.

PITG_21645.2, an effector of Phytophthora infestans isolates Heilongjiang (HLJ), could inhibit the hypersensitive response induced by INF1 in Nicotiana benthamiana. To identify that it is one of important effectors, the homologous genes of PITG_21645.2 were cloned from 10 isolates of P. infestans and three other oomycetes species including Phytophthora capsici, Phytophthora sojae and Phytophthora parasitica. They shared 93% to 100% identity with each other for the amino acid sequence. After co-infiltration with INF1, only PITG_21645.2^MP903,which contains the Serine differentiated to Asparagine of those homologues at the position of 31, couldn’t suppressed the HR triggered by INF1. And the site directed mutagenesis of the 31^th amino acid of PITG_21645.2^MP903 from Serine to Asparagine could restored the inhibition function. These implied that the 31^th position is one of the key sites to suppress the INF1-triggered HR. Meanwhile, we identified that PITG_21645.2^HLJ but not PITG_21645.2^MP903could also suppress the HR triggered by BAX, or other two secreted peptides PsojNIP and Avh238 of P. sojae, in N. benthamiana. Moreover, the transient expression of PITG_21645.2^HLJ could also enhance the susceptibility to P. infestans in N. benthamiana. It indicates that PITG_21645.2 plays important roles during the infection process for P. infestans.

Botrytis cinerea can infect more than 1 400 species of plants and cause serious losses around the world. Mito-TTP is a class of conserved proteins that are mainly responsible for transferring tricarboxylate substances in or out of mitochondria. It catalyzes obligatory exchanges of the dibasic form of a tricarboxylic acid for another tricarboxylate/H⁺, a dicarboxylate or phosphoenolpyruvate in Saccharomyces cerevisiae. In this research, the Mito-TTP gene was disrupted to study its biological roles in B. cinerea. The resulting mutants showed no difference in fungal growth compared to the strain B05.10, but displayed a number of phenotypic changes including reduced levels of conidial production and fungal virulence, the delayed development of sclerotia, and the increased sensitivity to CH₃COONa supplemented in growth media. All of these phenotypes could be rescued by complementing the mutants with the BcMito-TTP gene. The results suggested that BcMito-TTP is involved in the process of conidial production, sclerotial development, and the virulence of pathogen. The mechanisms are still under investigation.

Xanthomonas oryzae pv. oryzicola depends on Type III secretion system (T3SS) to cause disease in host plant rice. T3SS is encoded by hrp (hypersensitive response and pathogenicity), hrc (hrp conserved) and hpa (hrp associated) genes. Mutations of essential hrp or hrc genes usually abolish the bacterial ability to cause pathogenicity in host rice and hypersensitive response (HR) in non-host tobacco. The hrpD5 gene is the fifth gene with unknown function located in the hrpD operon of the hrp gene cluster. In this work, we constructed a deletion mutant of hrpD5 in Xoc strain RS105 (named as RΔhrpD5). Inoculation assays showed that RΔhrpD5 was not able to cause pathogenicity in host rice and failed to trigger HR in non-host tobacco. The lost functions were partially restored in the complementary strain CRΔhrpD5 which had reduced virulence on the susceptible rice and caused delayed HR on non-host tobacco compared to the wild type. Transcriptional analysis showed that mutation of hrpD5 affected the expression of downstream genes hrpD6, hrpE and hpaB in the hrpD operon and genes hrpF and hrpB1 in the HrpX operon, and led to decreased level of hrpX mRNA, but didn′t affect the promoter activity of hrpX. The yeast two-hybrid analysis revealed that HrpD5 interacted with the N terminal of HrpF. These results indicate that hrpD5 not only is essential for the pathogenicity of Xoc, but also regulates the expression of hrpX, the major hrp regulatory gene. Further functional investigation of HrpD5 may provide some clues to elucidate the roles of T3SS in pathogenicity of Xoc.

The rice fungal disease of sheath blight caused by Rhizoctonia solani not only leads to huge annual yield losses but also impairs the grain quality. In the present study, we evaluated the biocontrol potential of two Trichoderma harzianum strains 3S1-13 and 4S2-46 and their growth-promotion efficacy on rice. The results indicated that these two T. harzianum strains could not produce conidia on PDA medium unless presence of 1% yeast extract. Compared to the control treatment, the fermentation broth of 3S1-13 and 4S2-46 significantly inhibited mycelia growth and sclerotium formation of R. solani, suggesting these two strains are potential biocontrol agents against rice sheath blight disease. Our further study suggested the biocontrol efficiency of fermentation broth was better than that of conidial suspension. In addition, treatment with fermentation broth of strains 3S1-13 and 4S2-46 also enhanced the seed germination rate, root length and fresh weight of rice in comparison to control. In summary, our results suggest that T. harzianum strain 3S1-13 and 4S2-46 could be recognized as a useful resource for biocontrol agents against sheath blight and growth enhancer of rice.

The objectives of this study were to explore the pathogenicity of Fusarium semitectum causing root rot of alfalfa seedlings, to access the disease resistance and tolerance levels of alfalfa varieties, and to provide scientific basis for variety selection and its disease control. Fourteen varieties of alfalfa were inoculated with 9 pathogen isolates isolated from roots of 9 alfalfa varieties from 4 locations. The relative root length, relative seedling height, relative germination rate and disease index were assayed at 14 days after inoculation. The results indicated that : relative root length, relative seedling height, relative germination rate and disease index differed highly significantly among the 14 alfalfa varieties and 9 pathogen isolates (P<0.05). The isolate from ‘THG-1’ exhibited the strongest pathogenicity, while the isolate from ‘Aohan’ showed the weakest pathogenicity. The isolates from ‘WL-323HQ’, ‘Sitel’ and ‘Siriver’ exhibited strong pathogenicity, while the isolates from ‘Reindeer’,‘Legacy’ and ‘LM 400’ showed weak pathogenicity. Alfalfa variety ‘Algonguin’ showed the strongest resistance, while ‘Phabulous’ exhibited the weakest resistance. ‘Verna’, ‘Victoria’, ‘Hunter river’ and ‘Amerimultileaf’ showed strong resistance, while ‘WL-323HQ’, ‘Sanditi’, ‘Sandy’, ‘Amerilea721’ exhibited low-level resistance.

Surfactin is one of the peptides produced by Bacillus subtilis, which showes strong hemolytic activity and oil discharge capability. It has been confirmed that surfactin plays an essential role in biofilm formation and root colonization of B. subtilis strains. In this study, the effect of PhoR/PhoP two-component system on surfactin production in B. subtilis strain NCD-2 was investigated. In the hemolytic activities assay, the hemolytic activities in the phoR-null and phoP-null mutants decreased in the low phosphate medium (LPM) as compared to strain NCD-2 wild type. We found that wild type strain NCD-2 showed strong oil discharge capability, but not for phoR-null and phoP-null mutants in LPM. The biofilm formation capabilities were respectively reduced about 2.0 fold and 2.2 fold in phoR-null and phoP-null mutants compared to that of wild type strain NCD-2 in LPM. The surfactin could be detected from the lipopeptide extracts of NCD-2 by fast protein liquid chromatography (FPLC). Compared to wild type strain NCD-2, surfactin production in the phoR-null and phoP-null mutants reduced about 2.3 fold and 6.4 fold respectively in LPM but not in high-phosphate medium (HPM). Quantitative reverse transcription PCR (RT-qPCR) further demonstrated that the surfactin synthetase gene-srfAA was positively affected by phoR gene and phoP gene, and the expression of srfAA in LPM was reduced about 2.1 fold in phoR-null and phoP-null mutants compared to that of wild type strain NCD-2. All of these characteristics can be restored by complementation of the intact phoR gene and phoP gene in the mutants, respectively. Results indicated that the PhoR/PhoP two-component system positively regulates the surfactin production in B. subtilis strain NCD-2.

In order to understand the pathogenic mechanism of Me3 virulent population from Meloidogyne incognita and to provide the theoretical support for control strategy, the chemotaxis and infection of Me3 virulent and avirulent populations to resistant and susceptible host plants were analyzed. The chemotaxis potential of M. incognita populations on resistant pepper cultivar HDA149 and susceptible Qiemen were explored through analyzing the number of second-stage juveniles (J2) attracted by the root tip of pepper on the medium of water agar gel. Quantitative RT-PCR (q-PCR) was used to confirm the number of virulent population and avirulent population invading the host roots, and according to the result of q-PCR, we can directly estimate the infection ability of virulent and avirulent populations on host plants. The results showed that the numbers of J2s from virulent population gathered around the root tips of the susceptible and resistant pepper were both significantly higher than that from the avirulent population at different post-inoculation time. Furthermore, the analysis of the selective ability of M. incognita to host plants revealed that the numbers of J2s from virulent and avirulent populations gathered around the root tip of the Qiemen pepper were both significantly higher than that of the HDA149 pepper at different post-inoculation time.Based on the results of q-PCR, we can see that it is easier for virulent population infecting the host plant roots compared with avirulent population. In summary, the chemotaxis and infection potential of J2s from Me3 virulent population of M. incognita to root of host plants was stronger than that from the avirulent population,and the selectivity of J2s from both populations to the susceptible host plant were stronger than that to the resistant host plant.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropic race 4 (Foc TR4), is a typical vascular and soil-borne disease which has significantly threatened the sustainable development of banana industry. In order to reveal the infection process and pathogenesis of Foc TR4, the young mycelia (66.7 mg·mL^-1) of wild-type strain of Foc TR4 (WT-Foc TR4) cultured for 18-20 h were lysed with enzyme mixture for protoplasting, which consisted of 25 mg·mL^-1 driselase, 0.4 mg·mL^-1 chitinase, 15 mg·mL^-1 lysing enzyme and 1.2 mol·L^-1 potassium chloride. The resulted protoplasts of 2×10⁷cells·mL^-1 were used to test the efficiency of transformation mediated by polyethylene glycol, and up to 9 transformants per μg DNA were obtained. AmCyan, RFP and YFP gene were stably transferred into WT-Foc TR4, separately, using the protoplasts transformation system. The gene FoOCH1 encoding α-1,6-mannosyltransferase in WT-Foc TR4 was knocked out using the split-marker recombination technology. The genetic transformation and gene knockout system in this pathogen laid the foundation for the study of functional genomics and plant-pathogen interactions.

The gene MoHYR1 is important for full virulence and H₂O₂ tolerance in the rice blast fungus Magnaporthe oryzae. MoHYR1 was obtained by PCR with the template of M.oryzae cDNA and cloned into a prokaryotic expression vector pHAT2. The protein MoHYR1 was successfully expressed in strain Escherichia coli BL21(DE3) with an estimated molecular mass of 20 kDa. The protein was then purified by affinity chromatography and gel filtration. Finally, high purity of MoHYR1 was obtained, which contributes to further study on its structure and function.

Fusarium oxysporum f. sp. vasinfectum (Fov), the pathogen of cotton fusarium wilt, was discriminated into eight races and an Australian genotype in the world based on their pathogenicity to differential hosts. The races of 3, 7 and 8 had been reported in China, but a suspected novel genotype of Fov was frequently identified in our lab. The purpose of this study was to develop a PCR-based technique for fast detection of the novel genotype strains from large number of samples collected in various cotton-growing areas. By performing gene sequence alignment of elongation factor (EF-1α) between races 3, 7, 8 and the novel genotype strain, several new genotype strain-specific bases were obtained. A pair of novel genotype strain-specific primers was designed based on those specific bases and used for PCR detection. It was confirmed that the primer set was specific for the novel genotype strains. Seventy seven Fov isolates collected from the main cotton-growing areas in Hebei province were tested with the novel genotype strain-specific primers, and two suspected isolates were obtained. The phylogenetic tree analysis revealed that these two suspected isolates formed a single clade with the novel genotype strain and showed a closer genetic relationship to the Australian genotype strain than the other races based on the combined EF-1α and beta-tubulin genes sequence data. It was concluded that the new method could be used for rapid detection of the novel genotype strain of Fov.

Citrus fruit decay caused by fungi during late stages of fruit growth, postharvest transit and storage has been very common. The suspected fungal pathogen isolated from brown and hard fruit in a citrus orchard in Ganzhou, Jiangxi Province in 2016 was identified as Botryosphaeria dothidea based on the morphological and molecular characteristics and pathogenicity test. The pathogen could also infect twig or branch, trunk and fruit of apple. To our knowledge, this is the first report of B. dothidea infecting citrus.

A leaf blight disease on pumpkin(Cucurbita moschata)occurred in Leizhou, Guangdong. The water-soaked lesions appeared at the edge of the leaves of infected-pumpkins, and gradually formed large lesions. The nearly-circular water-soaked lesions also appeared on leaves with yellow haloes. The petioles and stolons were also infected by the pathogen and exhibited water-soaked rot. A bacterium was isolated from the diseased spots. The colonies on KB medium were elliptical, milky white, translucent with uneven edges, and produced fluorescence under ultraviolet light. Pathogenicity test showed that the bacterium could infect 6 varieties of pumpkin and cause leaf blight symptoms being similar to those in the fields. The physiological and biochemical analysis showed that characteristics of the bacterium were in accordance with Pseudomonas syringae pv. syringae. PCR amplification with Pseudomonas specific primers Ps-for/Ps-rev and P. syringae pv. syringae specific primers Group III-F/Group III-R produced the expected 1018 bp and 750 bp fragments, respectively. An expected 750 bp product encoding the syringomycin was amplified from the isolates of the bacterium with P. syringae pv. syringae syrB gene specific primers B1/B2. Both of the phylogenetic analyses based on the 16S rDNA and gyrB gene sequences showed that the bacterium isolates were very nearly related to the P. syringae pv. syringae isolate HS191 (CP006256) and they clustered in one branch. By artificial inoculation, the bacterium could also infect zucchini, loofah, eggplant, tomato, common bean and hyacinth bean. These results reveal that the pathogen of bacterial leaf blight disease on C. moschata in Guangdong is P. syringae pv. syringae. This is the first report of P. syringae pv. syringae causing bacterial leaf blight on pumpkin in China.

Rice false smut, caused by Ustilaginoidea virens (U. virens), is a ubiquitous disease in rice planting areas worldwide. In response to U. virens infection, the accumulation of reactive oxygen species (ROS) was significantly increased in rice. The infection of U. virens suppressed rice pollen germination and pistil development, and led to production of a large number of false smut balls and bleached grains. To detect the ROS in healthy, infected and bleached spikelets, H₂O₂ was stained and visualized by exposing to DAB. The absorbance of oxidized DAB at 465 nm was measured, and a significant increase in infected spikelets solution was detected compared to the healthy spikelets. After DAB treatment, brown oxidized DAB precipitates could be detected readily on the position of U. virens in infected spikelets but not on the mock-infected spikelets. DAB oxidation seemed to be associated with locations colonized by the fungus at spikelets.

The cluster of Cop genes is the main locus to control the copper resistance in Xanthomonas species. And the PXO99, a wild strain of Xanthomonas oryzae pv. oryzae (Xoo) causing the bacterial blight in rice, is more sensitive to copper than other wild strains. In this study, we identified that the deletion of six amino acids on CopAB genes is the crucial factor of copper ion sensitivity in PX099. After testing copper resistance ability and pathogenicity with CopAB knock out mutant or the complementary strains, we found that CopAB was not only involved in copper tolerance, but also acted as a pathogenic factor in Xoo. Alternatively, we screened out four copper sensitive strains which contain the similar mutation on CopAB from domestic strains. The CopAB complementary PXO99 restored the copper tolerance, as well as the 3 out of 4 wild copper-sensitive strains without PthXo1, failed to overcome the xa13-mediated resistance that caused the similar disease symptoms on both IR24 and IRBB13. These results suggest that the copper sensitivity of PXO99 is not associated with xa13-mediated race-specific resistance.

Complete sequence of one Lily mottle virus (LMoV) isolate which mainly infected lily from Songming county of Yunnan province was obtained and analyzed. Sequence comparison of coat protein (CP) gene among four LMoV isolates from Songming (LMoV-SMi1, LMoV-SMi2) and Yuxi (LMoV-YXi1, LMoV-YXi2) showed different identities at nucleotide and amino acid sequence levels, which could be divided into two subgroups, LMoV-YXi1 and LMoV-YXi2 belonged to subgroup I, while LMoV-SMi1 and LMoV-SMi2 belonged to subgroup II. The two groups shared 86.7%-89.5% and 90.1%-92.7% identity at nucleotide and amino acid sequence levels, respectively. Sequence analysis of the cp gene revealed that there was a three-nucleotide deletion in the two Yuxi isolates compared with that of the two Songming isolates. Amino acid sequences comparison of the cp gene of LMoV isolates available in GenBank showed that all of the isolates could be divided into two subgroups, and almost all of the isolates in subgroup I including a threonine deletion compared with those in subgroup II. The results indicated that all of the analyzed LMoV isolates could be divided into two populations. The related properties and spatial structure of the CP of LMoV-SMi2 were predicted, and the result showed that the CP of LMoV-SMi2 was spherical with great surface probability plot and no transmembrane domain was detected. The antigenic determinant mainly located at aa12-22, aa31-42, aa83-99, aa179-191, aa215-223, aa249-259, and these regions could be used for antibody preparation. The CP spatial structure between LMoV-SMi2 and LMoV-YXi1 isolates were similar though there were some differences in their predicted CP secondary and tertiary structures.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is the most devastating wheat disease in China. Wheat growing region of Sichuan Province plays a key role for stripe rust spread and epidemic in China. To evaluate the resistant level and detect the stripe rust resistance gene(s) of commercial wheat cultivars and advanced lines in Sichuan Province, 100 wheat cultivars (lines) collected from Sichuan Province were inoculated with Chinese Pst races G22-83, CYR32 and CYR33 at the seedling stage, and the genomic DNA of these cultivars (lines) were tested with the closely linked markers of all-stage resistance genes Yr5, Yr9, Yr10, Yr15, Yr26 and adult-plant resistance gene Yr18. The disease evaluation results indicated that 58%, 63% and 43% wheat cultivars (lines) showed resistance to CYR32, CYR33 and G22-83 in the seedling tests, respectively. Among these, 28 cultivars (lines) were resistant to all three Pst races. Molecular detection suggested that the resistance genes Yr9, Yr10, Yr15 and Yr26 were found in 24%, 9%, 5% and 26% cultivars (lines), respectively. There was no indication of Yr5 and Yr18 in any cultivar (lines).

Rice kernel smut pathogen attacking primarily male sterile lines, is the main pathogenic factor that affects the yield and quality of hybrid rice seed reproduction in southern of China. In order to excavate rice varieties resistant to kernel smut, and provide resistant rice sterile materials for breeding, seventy-eight male sterile rice lines from Hubei, Fujian and Sichuan were evaluated at the heading period by inoculating with Tilletia horrida during 2014-2016. The results showed that the most of the seventy-eight lines were susceptible, but four of them were resistant to rice kernel smut pathogen. Among them, 4766A was the first cultivar highly resistant to rice kernel smut in China.

The objective of this study was to evaluate the pathogenicity of Fusarium acuminatum isolates to various alfalfa varieties. Fourteen varieties of alfalfa were inoculated with 15 F. acuminatum isolates obtained from alfalfa roots collected at 4 locations. The relative root length, relative seedling height, relative germination rate and disease index were assayed at 14 days after inoculation. The results indicated that the root length of alfalfa seedlings was significantly reduced by all of the 15 isolates (P<0.05). Overall, the pathogenicity of each isolate varied among the 15 isolates. The isolate from ‘WL-323HQ’ exhibited the strongest pathogenicity, while the isolate from ‘WL-525HQ’ (planted in experimental area of Neihuang County of Anyang City) showed the weakest pathogenicity. The isolates from ‘WL-525HQ’ (planted in experimental area in Zhengzhou City), ‘Legacy’ , ‘Affinity’ and ‘Millionaire’ showed strong pathogenicity, while the isolates from ‘Aohan’, ‘Farmers’, ‘WL-323ML’ and ‘Dingdian’ showed weak pathogenicity. The disease resistance of the 14 alfalfa varieties also varied, among of them, ‘Amerimultileaf’ showed the strongest resistance. ‘Amerileaf721’, ‘Victoria’, ‘Powerplant’ and ‘Legacy’ exhibited high-level resistance. ‘Sandy’, ‘Phabulous’, ‘Hunter river’ and ‘Alfaking’ showed low-level resistance. ‘Vernal’ had the weakest resistance.

Wheat stripe rust were monitored using hyper-spectrometer and UAV aerial photography in the field, respectively. The relationships among disease index and hyperspectral canopy reflectance parameters or UAV digital image parameters were analyzed. The results showed that there were significantly correlations between disease index and the hyperspectral parameters (DVI, NDVI, GNDVI) or UAV digital image color feature parameters (R, G, B). In general, the correlations between hyperspectral parameters and disease index were higher than those between UAV digital image parameters and the disease index. Furthermore, hyperspectral parameters DVI, NDVI, GNDVI were negatively and significantly correlated with UAV digital image parameters R, G, B. We constructed the estimation models of wheat stripe rust based on hyperspectral parameter GNDVI and UAV digital image parameter R, respectively, both the models fitted well, and the model based on GNDVI performed better than the model based on R, however, UAV digital images have the advantages of undertaking fast detection in large area, so in practice, we can choose one methods or one of the parameters to estimate the occurrence and epidemic of wheat stripe rust in the field as needed.

Strawberry gray mold caused by Botrytis cinerea Pers. is an important infectious disease, which can cause fruit rot of strawberry and gives a great threat to fruit yields and post-harvest fruits of strawberry. To study the epidemics of strawberry gray mold in greenhouse and to investigate the corresponding influence factors, the number of airborne conidia of B. cinerea, the rate of petals infected by the fungus and the number of new diseased fruits were dynamically monitored in the growing seasons of 2013 to 2014 and 2014 to 2015, respectively. Combining with meteorological factors recorded in the greenhouse, all collected data were analyzed. The results showed that the airborne conidia concentration of B. cinerea was high from 5:00 to 18:00, especially during 11:00 to 14:00 in one day. The analysis results based on the obtained data in the two growing seasons of strawberry demonstrated that the hourly number of trapped conidia had highly significant positive correlation with temperature and light intensity (P≤0.01), and had highly significant negative correlation with relative humidity (P≤0.01). The number of new diseased fruits had highly significant positive correlation with both the number of trapped conidia (r=0.872, P≤0.01) and the infection rate of the fresh petals (r=0.807, P≤0.01) on the seventh day before the corresponding fruit survey, indicating that this number of trapped conidia and this infection rate could be used as references to predict strawberry gray mold seven days in advance. The results of this study are helpful to understand the occurrence and influence factors of strawberry gray mold in greenhouse, and have provided some basis for the prevention and control of this disease.

Heterodera filipjevi is one of the important pathogenic nematodes that endanger the cereal crops in temperate zone, and it caused serious influence of the yield and quality. H. filipjevi is a newly identified species of cereal cyst nematode in central China. More than three years studies showed that Xuchang population is a new pathotype of H. filipjevi, and its pathogenicity was stronger than other populations of cereal cyst nematode in Huang-Huai Plain. This study aims to establish a rapid and accurate molecular detection system of Xuchang population, lay the foundation for monitoring and controlling Xuchang population of H. filipjevi and provide the basis for breeding and utilization of resistant varieties. This study focused on using RAPD-SCAR technique to analyze 9 CCN populations of 5 different CCN pathotpyes from Huang-Huai Plain. Moreover, 8 extra CCN populations were added to verify the specificity and validity of SCAR markers. 331 random primers were screened and two random primers with RAPD method were found related to Xuchang population, Primer S86, S178 could product polymorphic bands of Xuchang population about 550 bp and 1 200 bp respectively. The two RAPD markers related to Xuchang population of H. filipjevi had been transformed into SCAR markers successfully. The results of the specific detection showed that the two SCAR markers can detect the Xuchang population from other CCN populations in Huang-huai floodplain. The two SCAR markers can be used to detect and identify H. filipjevi Xuchang population.

Analysis and evaluation the pathogenicity of Verticillium dahliae Kleb. is essential for breeding of disease-resistant cotton plants and integrated control against Verticillium wilt. In this paper, we focus on the calibration of disease index in different batch tests and the standard of classification on virulence patterns of Verticillium dahliae based on 167 isolates collected from three major cotton growing regions in China. We found a set of strain and variety, the intermediate virulent strain Vd076 as the reference strain and the Jimian 11 with stable susceptibility as the corrected differential host facilitated the dataset compatibility after correction of the different batch tests data when the disease index of Jimian 11 reaches 50.0±5.0 by infection of Vd076. Based on the phylogenetic analysis, the criteria of classification for pathogenicity of V. dahlia were set up in which the average of the corrected disease indexes for three patterns of strains with high virulence, moderate virulence and low virulence were﹥40.0, 20.1~40.0 and 20.0, respectively. The analysis of different groups of differential hosts supported that Zhongmiansuo 41, Yumian 21, Lumianyan 28, Zhongmiansuo 35, Zhongmiansuo 8 and Jimian 11 were to be the differential hosts. This study provides the technical support for the virulence identification and variation of V. dahliae.

Bakanae is an important disease of rice across all major rice-growing regions in China leading to serious rice yield losses. Here we describe a loop-mediated isothermal amplification (LAMP) assay on direct detection of F. andiyazi in the diseased rice tissues, for rapid diagnoses of rice bakanae caused by this pathogen. Based on the sequence of trichothecene 3-O-acetyltransferase (TAT) gene in F. andiyazi, a set of species-specific primers was designed and screened to generate a positive color (sky blue) in the presence of F. andiyazi with the addition of hydroxynaphthol blue (HNB) indicator prior to amplification, whereas no color change (purple) in the negative reaction tubes of other species isolates under isothermal condition at 64℃ for 80 min. Using the TAT-Fan-LAMP assay with the detection limit at 100 pg·μL^-1 genomic DNA for F.andiyazi, we successfully and rapidly diagnosed the suspected diseased rice plant samples collected from Jiangning, Nanjing and Jurong, Zhenjiang in Jiangsu Province. This method can be used as a new way of diagnosing rice bakanae caused by F. andiyazi.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is one of the most destructive seed-borne diseases of watermelon and other cucurbits. This seed-borne quarantine disease has been reported in most watermelon and melon producing regions worldwide. Since the infected seeds are the most important primary inoculum source of BFB, seed health testing (SHT) and subsequent elimination of the contaminated seeds is one of the most effective methods to control the disease. Bio-PCR is widely used in SHT for the detection of A. citrulli. As A. citrulli has abundant genetic diversity and many other closely related species, specific and sensitive primers are very important for the success of the seed health testing. To screen the best primers for detection of A. citrulli by Bio-PCR, 33 bacterial strains including 17 strains of A. citrulli, 10 strains of other Acidovorax species and 6 strains of other genera of plant pathogenic bacteria were chosen to evaluate 7 pairs of primers reported so far. Results indicated that the primer pair SEQID4^m/SEQID5 had the best specificity that could distinguish all the tested strains of A. citrulli from those of other species and genera. The sensitivity of this primer pair was further tested with the Bio-PCR assay of A. citrulli. Results showed that the detection limit of the assay was 10² CFU·mL^-1 for the pure bacterial culture, while for watermelon seeds the detection limit were 0.01 CFU·g^-1 and 0.1 CFU·g^-1 when seed extracts were cultured on semi-selective media ASCM and EBBA, respectively.

A new fungal disease of stem wilt was found on Polygonum capitatum. The isolate was obtained from diseased field sample. This study was to identify and characterize the isolate. Based on analysis of ITS, EF-1α and β-tubulin sequences, the isolate was placed in Fusarium equiseti clade in phylogenetic tree. In combination of morphological characteristics the fungal isolate was identified as F. equiseti, a new pathogen on Polygonum capitatum. To determine the optimal nutritional and cultural conditions for the pathogen, the biological characteristics test was conducted. The results showed that the optimal carbon and nitrogen sources were fructose and sodium nitrate, respectively, and the mycelia was not sensitive to light with the optimum growth temperature was at 28℃ and favorable pH value at 7, while the lethal temperature for mycelia growth was 65℃ for 10 minutes.

Lr26 and Lr19 are two known important wheat leaf rust resistance genes. The positive and negative markers of the two genes have been developed at present. A total of 17 commercial wheat cultivars collected from Sichuan province were inoculated with 14 Chinese Puccinia triticina races for gene postulation in the greenhouse and used to detect Lr26 and Lr19 by multiplex PCR, respectively. The results showed that Lr26 was detected in six wheat cultivars, and no cultivar was found to carry the Lr19 gene. The multiplex PCR can be widely used for marker-assisted selection of Lr26 and Lr19.

Gummy stem blight,usually seen on the stems, leaves and fruits, is one of the most serious threats to production of balsam pear (Momordica charantia L.) in Guangxi with reduction of the quality and heavy losses in yield. The pathogenic organisms of the disease were still obscure because several fungal species with most similar morphology were reported to be the causative agents. In this paper, the isolate in imperfect stage from the samples of gummy stem blight of balsam pear in Guangxi was identified as Stagonosporopsis cucurbitacearumbased on the morphological characteristics, inoculation experiments and phylogenetic analysis with rDNA-ITS sequences.

Papaya ringspot virus (PRSV) is a major limiting factor to cucurbit production worldwide. One zucchini sample showing symptoms of leaf mosaic and fruit ringspot was collected from Ji’nan city of Shandong province. Primary experiments showed that the sample was infected with PRSV, which was designated as PRSV-SD accordingly. The complete genomic fragment of PRSV-SD was obtained with RT-PCR. The results of sequencing showed that the full genomic sequence of PRSV-SD was 1 0337 nucleotides (nt) excluding the 3′- terminal poly (A) tail. The 5′- and 3′-untranslated regions (UTR) were 90 and 206 nt, respectively. The putative polyprotein was 3 346 amino acids in length. Comparison of the PRSV-SD isolate with 18 other PRSV isolates revealed that they shared nucleotide identities of 82.1%~89.3% at the complete genomic levels and amino acid identities of 90.6%~94.7% for the polyprotein. No recombination was detected throughout the genome of PRSV-SD. Phylogenetic analysis with complete genomic sequences indicated that the PRSV isolates were clustered into two major groups: Asia and America and PRSV-SD was clustered to the Asia group. Selection pressure analysis revealed that all of the 11 proteins of PRSV-SD were under negative selection, but positive selection sites were detected in P1, P3, 6K1, NIa-pro and NIb. Our research results provide a theoretical foundation for the detection and prevention of PRSV. Key words:Papaya ringspot virus; complete genomic sequence; phylogenetic analysis; selection pressure; recombination 中图分类号:S436.429; Q939.46 文献标识码:A 文章编号:0412-0914(2018)02-0285-04 番木瓜环斑病毒(Papaya ringspot virus,PRSV)属于马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)^[1]。根据寄主范围可划分为P和W 2个株系,其中P株系侵染番木瓜(Carica papaya L.)和葫芦科(Cucurbitaceae)作物,而W株系只侵染葫芦科作物,两者血清学反应密切相关。PRSV可引起植株叶片褪绿、花叶、卷曲等症状,在果实表面形成环斑。    PRSV于20世纪40年代末在美国首次报道,目前该病毒在巴西、印度、波兰等国均有发生^[2]。2000~2001年,PRSV使我国海南番木瓜损失严重。2005年以来,我国广东、四川先后检测到PRSV侵染罗汉果、苦瓜等葫芦科作物^[3]。2016年从山东西葫芦上检测到PRSV,该分离物属于W株系^[4]。我国关于PRSV进化的研究大多集中于P株系,有关W株系全基因组序列分析的报道相对较少。为了进一步研究我国PRSV-W株系的基因组特性,为PRSV的监测预警提供依据,我们测定了PRSV山东分离物的全基因组序列,并进行了核苷酸和氨基酸序列一致率、重组、系统发育和基因选择压力分析。 2期黄显德,等:番木瓜环斑病毒山东分离物的全基因组序列分析      植物病理学报48卷 1 材料与方法 1.1 材料 发病西葫芦样品采自山东省济阳县。大肠杆菌DH5α由本实验室保存。植物总RNA提取试剂盒TRIzol、DNA凝胶回收试剂盒等均购自北京全式金公司;Taq DNA聚合酶、 pMD18-T克隆载体等购自TaKaRa公司;SuperScript^TM Ⅳ逆转录酶购自Invitrogen公司。其他生化试剂及普通化学试剂均为进口或国产分析纯。 1.2 实验方法 1.2.1 扩增策略及引物合成 根据GenBank中PRSV基因组序列,设计引物分4段扩增基因组序列。根据所得序列设计5′-RACE引物扩增5′-端片段。测序后用SeqMan拼接得到全基因组序列。所用引物详见表1。 1.2.2 植物总RNA提取及RT-PCR扩增 按照RNA提取试剂盒说明书进行植物总RNA提取。以总RNA为模板,用随机引物反转录合成cDNA。通过4次PCR和5′-RACE扩增PRSV-SD基因组片段。 1.2.3 克隆及序列测定 电泳分离PCR产物,切胶回收后连接到pMD18-T载体上,转化E. coli DH5α感受态细胞,挑选阳性克隆进行核苷酸测序。

Meloidogyne graminicola is one of the most dangerous plant parasitic nematodes in rice and a major problem restricting rice production in China and Asia. M. graminicola has a broad host range and can infect rice plants in highland, lowland and deepwater. This nematode uptakes nutrients from giant cells and form hook-like root galls. The secretions from esophageal directly cause harm to rice and negatively affect metabolism and immunity of host plant cell. To date, chemical nematicides are often used in nursery bed to control this nematode and few rice varieties are found to be resistant to M. graminicola. In the present study, host range, biology, etiology, pathogenic mechanism and control methods of M. graminicola are reviewed. The control strategies using genetic modified technology such as gene edit to effectively control this pest are discussed, which will provide a theoretical basis for the prevention and controlling of rice root-knot nematode in China.

In order to confirm the pathogen of kernel smut on rice seeds and its pathogenicity in the hybrid rice seed production areas of south China, the isolates were obtained from the samples of rice seeds with kernel smut, collected from Anhui, Hunan, Jiangsu, Guizhou, Suining, Xinjin, and Wenjiang in August 2015, by culturing spore suspension. The following characterizations were conducted according to the morphological characteristics, nucleus fluorescence staining, and rDNA-ITS sequence analysis. Meanwhile, the pathogenicity test of the pure cultures from the infected rice tissues was performed according to Koch’s postulates. The results showed that the thirty-five strains exhibited no significant differences in morphology. The teliospores in shape was sphere or near round, and the secondary microspore were liner, curve and mononucleate. The results of rDNA-ITS sequence analysis showed that 7 isolates were 100% identity with Tilletia horrida DQ827699. The pathogenicity tests showed that all the strains could cause the typical symptoms on rice seed of rice kernel smut with different level of pathogenicity, in which the JS strain from Jiangsu reached the highest level of pathogenicity and conidial germination rate leading to the highest losses in yield and amount of infected seeds per spike. The optimal temperature for mycelial growth of the seven test strains were 28℃ while light did not obviously affect mycelial growth.

The total of 39 isolates were collected from peanut plants with southern stem rot symptoms in 12 provinces of China from 2013 to 2016. All the isolates belonged to Sclerotium rolfsii based on the ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences, and were classified into two groups in which one contained 22 isolates and the other contained 17 isolates. The 23 MCGs (mycelial compatibility groups) and related biological characteristics were determined for the 39 isolates, in which 8 MCGs contained 2 to 6 isolates and the others contained only one isolate. The colony morphology of strains in the same mycelial group was similar, and the mycelial growth rate and sclerotia size among most strains were similar, but the individual strains were different. All the isolates could produce oxalic acid and exhibited significant differences in colony morphology, growth rate, sclerotia size and weight with the growth rate from 0.47 to 2.32 cm·d^-1, the sclerotia diameter from 0.71 to 1.87 mm and dry weight for single sclerotia weight from 0.24 to 2.64 mg.

Phoebe bournei, one of the national second-class endangered plants, is the excellent precious timer species with broad market prospect. Leaf spot was newly found leaf disease, which resulted in stress and abnormal growth of plants in the extension of Ph. bournei plantation. The causing agent of the disease was identified as Pestalotiopsis microspora by morphological characteristics and rDNA-ITS sequences analysis. The result of the biological test showed that the optimal conditions for mycelia growth of Pe. microspora strain ZZ-03 were Czapek agar or PDA+PBL(pH 6.0)at 28℃ in dark. The spore production was optimal on PDA medium(pH 8.0)at 28℃ in dark. Conidia germination was positively correlated with relative humidity at 28℃ with pH 8.0 in dark. The best carbon and nitrogen sources for mycelia growth were glucose and beef extract, respectively, while glucose and peptone were the best for conidiation, respectively.