Peach shoot blight caused by Phomopsis amygdali has led to considerable economic losses in major peach fruit-growing areas. A biocontrol bacterium, Bacillus licheniformis W10 was successfully identified in our previous study. Furthermore, we found that B. licheniformis W10 could produce an extracellular antifungal protein that exhibited a strong inhibitory effect against many plant pathogens. However, the biocontrol potential of B. licheniformis W10 against peach shoot blight pathogen is still unclear. Therefore, the biocontrol effects of the B. licheniformis W10 bacterial suspension and its antifungal protein on P. amygdali were investigated. The results showed that the B. licheniformis W10 bacterial suspension and antifungal protein inhibit mycelial growth, sporulation, conidial germination and germ-tube elongation of P. amygdali. The bacterial suspension (2 × 109 cfu/mL) and antifungal protein at 1.5320 mg/mL completely inhibited mycelial growth. A 5 × 109 cfu/mL bacterial suspension and antifungal protein at 1.5320 mg/mL had 94.9% and 87.5% inhibition rates on the sporulation of P. amygdali, respectively. The bacterial suspension of 1 × 109 cfu/mL and antifungal protein at 6.4992 mg/mL completely inhibited the conidial germination and germ-tube elongation of P. amygdali. Furthermore, low concentrations of the bacterial suspension (5 × 108 cfu/mL) and antifungal protein (1.6248 mg/mL) still had an above 80% inhibition rate against conidial germination and germ-tube elongation. In addition, the bacterial suspension and antifungal protein were demonstrated to have destructive effects on the hyphal shape and structure of P. amygdali, with hyphal cell distortion, tumescence, protoplasm leakage, cell wall breakage, and hyphal rupture. Overall, our results suggest that B. licheniformis W10 can be used as a potential biocontrol agent for the management of peach shoot blight.

树生黄单胞菌李致病变种Xanthomonas arboricola pv.pruni (Xap是一种危害桃树的植物病原细菌,会导致桃树发生细菌性穿孔病.本文主要对该细菌性穿孔病菌的分布、命名、遗传多样性、致病机理和防治技术的研究进展进行了总结,重点阐述了桃细菌性穿孔病的不同防治方法,以期能提供新的防治思路,提升防治水平,保障桃果的安全生产.

[Background] Brown rot on peach and nectarine trees caused by Monilinia spp. is an important fungal disease, and produces serious economic losses frequently in China. There may exist more than one species of peach brown rot pathogens. [Objective] To clarify the species and pathogenicity of brown rot pathogens from different peach-growing areas in China. [Methods] The pathogens were isolated from peach brown rot fruits of different peach production areas, using the method of single spore isolation. Identification and analysis of isolates were carried out by morphological characteristics, molecular biology methods, and inoculation assay. [Results] In this study 15 brown rot pathogen isolates were obtained from 8 different peach-growing areas. The morphological characteristics of the isolates were slightly different from each other, but the isolate hfyn from Yunnan Province was significantly different from the other isolates. Based on its morphology, rDNA ITS sequence and phylogenetic analysis, the isolate hfyn was identified as Monilia yunnanensis, which was a newly found brown rot pathogen, and the other 14 isolates were identified as Monilinia fructicola. PCR amplification using several pairs of specific primers confirmed the morphological and rDNA ITS identification results. Inoculation tests showed that the pathogenicities on peach fruits of 15 isolates from different peach producing areas were different. The isolates from Lishui, Zhejiang Province, Shijiazhuang, Hebei Province, and Qingdao, Shandong Province had more virulent, and the virulences of isolates from Kunming, Yunnan Province and Tai'an, Shandong Province were relatively weak. [Conclusion] The results indicated that the major pathogen of brown rot in peach-growing areas in China was Monilinia fructicola, and there were differences in morphology and pathogenicity among these isolates from different peach-growing areas. The result from the present study may provide a scientific basis for managing peach brown rot epidemics.

[目的]筛选出地衣芽孢杆菌W10与化学农药毒力增效复配剂,明确复配剂对桃枝枯病的田间防效.[方法]采用菌丝生长抑制法,测定W10菌液与化学农药不同配比对桃枝枯病菌的联合毒力,并评价复配剂对桃枝枯病的田间防效.[结果]W10与咪鲜胺1∶1和1∶2复配对桃枝枯病菌有明显的增效抑制作用.3年田间小区试验表明W10和咪鲜胺1∶1复配对桃枝枯病防效达到或超过了咪鲜胺单剂.[结论]地衣芽孢杆菌W10可与咪鲜胺复配协同防治桃枝枯病,不仅提高生防比例和效果,还减少农药使用.

桃褐腐病是桃树上的一种重要病害,导致桃果经济损失严重.本文对不同产区桃褐腐病菌的生物学特性进行了研究.来源不同产区的桃褐腐菌株总体在PDA培养基、25℃、pH值6.0～7.0及每天光照12h条件下生长最好;菌丝生长最适碳源和氮源分别是淀粉和牛肉浸膏.褐腐菌株分生孢子平均在25℃、pH值7.0及每天光照12h条件下萌发率最高.不同产区褐腐菌株间存在生物学差异,表明病菌存在丰富的生理生化多样性.研究结果表明,不同的营养、温度、光照、pH值等环境条件对桃褐腐病菌菌丝生长和孢子萌发有显著影响.

Phytophthora cactorum, an oomycete pathogen, infects more than 200 plant species within several plant families. To gain insight into the repertoire of the infection-related genes of P. cactorum, Illumina RNA-Seq was used to perform a global transcriptome analysis of three life cycle stages of the pathogen, mycelia (MY), zoospores (ZO) and germinating cysts with germ tubes (GC). From over 9.8 million Illumina reads for each library, 18,402, 18,569 and 19,443 distinct genes were identified for MY, ZO and GC libraries, respectively. Furthermore, the transcriptome difference among MY, ZO and GC stages was investigated. Gene ontology (GO) and KEGG pathway enrichment analyses revealed diverse biological functions and processes. Comparative analysis identified a large number of genes that are associated with specific stages and pathogenicity, including 166 effector genes. Of them, most of RXLR and NLP genes showed induction while the majority of CRN genes were down-regulated in GC, the important pre-infection stage, compared to either MY or ZO. And 14 genes encoding small cysteine-rich (SCR) secretory proteins showed differential expression during the developmental stages and in planta. Ectopic expression in the Solanaceae indicated that SCR113 and one elicitin PcINF1 can trigger cell death on Nicotiana benthamiana, tobacco (N. tabacum) and tomato (Solanum lycopersicum) leaves. Neither conserved domain nor homologues of SCR113 in other organisms can be identified. Collectively, our study provides a comprehensive examination of gene expression across three P. cactorum developmental stages and describes pathogenicity-related genes, all of which will help elucidate the pathogenicity mechanism of this destructive pathogen.

The plant growth regulator paclobutrazol is widely used in agriculture to inhibit plant growth,promote tillering,increase plant yield and so on.In order to provide the basis for control peach shoot blight by paclobutrazol,this paper used the pathogen of peach shoot blight,Phomopsis amygdali,as test strain,and measured the peach shoot blight lesion size and the content of H2O2 and MDA in the shoots after spraying the paclobutrazol,using shoot inoculation in vitro.The results showed that paclobutrazol had the virulence to P.amygdali,with ECs0 value of 1.623 mg · L-1,and had a destructive effect on hyphal shape of P.amygdali,with hyphal cell distortion and tumescence.The 400,500,600,700 and 800 mg · L-1 paclobutrazol could control extension of disease spot on the twigs in vitro in different degrees.Among them,the inhibitory effect of the concentration of 600 mg · L-1 for paclobutrazol was the best,and the lesion size was reduced by 71.15％ and 69.70％,respectively,compared with the water control and methanol control.The 600 mg · L 1 paclobutrazol had a significant effect on reducing content of H2 O2 and MDA in P.amygdali infected twigs.Therefore,a certain concentration of paclobutrazol could reduce the pathogen damage to the host,and the inhibitory effect of paclobutrazol on pathogen infection may be related to the regulation of host disease resistance by increasing the activity of antioxidant substances.

为减少化学药剂的使用,明确生防细菌地衣芽孢杆菌W10与化学药剂复配对桃褐腐病菌Monilinia fructicola抑菌活性的增效作用,采用菌落直径抑制法,检测W10菌液和抗菌蛋白与化学试剂复配的抑菌效果.结果显示,W10抗菌蛋白与化学药剂复配增效显著,与多菌灵100:1增效作用最大(SR=4.32).证明生防细菌W10菌液及抗菌蛋白与化学药剂以合适比例复配对桃褐腐病菌的抑制有增效作用,可用于该病害的防治.

Two novel polygalacturonase (PG) genes, RsPG3 and RsPG4, were cloned from Rhizoctonia solani isolate YN-7, which is a member of anastomosis group (AG) subgroup 1A and causes rice sheath blight. The predicted protein product of RsPG3 contained 239 amino acid (aa) residues, with a molecular mass of 25.0 kD, whereas RsPG4 encoded a deduced protein of 345 aa residues, with a mass of 37.5 kD. Sequence alignment, phylogenetic analysis and gene ontology indicated that RsPG3 and RsPG4 had endo-PG and exo-PG activity, respectively. Recombinant RsPG3 and RsPG4 both exhibited PG activity that led to the destruction of rice sheaths and release of reducing sugars. Pathogenicity assays showed that the two PGs induced tissue necrosis in rice sheaths, indicating that both RsPG3 and RsPG4 are important virulence factors in the R. solani–rice interaction. Further studies are underway to more clearly define the role of these enzymes in rice cell wall degradation and their interaction with PG inhibitor proteins.

Rice sheath blight, which is caused by Rhizoctonia solani , is a devastating disease in many areas of the world where rice is cultivated. The role of polygalacturonase (PG) in the infection process of R. solani was investigated in the present study by cloning and investigating the function of a new PG-encoding gene, which was designated RsPG2 . Bioinformatics showed that RsPG2 consisted of a 1,633 bp open reading frame (ORF) with six introns; the deduced protein contained 436 amino acids with a predicted mass of 45.88 kDa after cleavage of the predicted 17-amino acid signal sequence. Culture supernatants obtained from Pichia pastoris producing recombinant RsPG2 degraded polygalacturonic acid in vitro, thus confirming that RsPG2 encodes a PG gene. Purified recombinant RsPG2 also degraded rice tissue and elicited obvious necrotic symptoms 48 h after inoculation. Real-time qRT-PCR indicated that the expression of RsPG2 was strongly induced during the infection of rice by R. solani . These data unequivocally demonstrate that RsPG2 plays an important role in R. solani infection and provide further insights for elucidating the function of pg genes in the virulence of R. solani .

[目的]明确生物和化学农药复配对桃流胶病菌的抑制效果.[方法]采用菌丝生长抑制法,测定10种生物药剂对桃流胶病菌的毒力,将毒力强的生物农药与化学农药丙环唑、多菌灵和咪鲜胺进行复配,探讨复配剂对桃流胶病菌的联合毒力效果.[结果]毒力作用最强的生物农药为梧宁霉素,其次是多抗霉素和申嗪霉素,EC枷值分别为0.6761、5.0119、6.9183 mg/L.多抗霉素、申嗪霉素与3种化学农药复配均有较好的增效作用,而梧宁霉素与多菌灵、咪鲜胺复配表现为相加或增效作用.[结论]生物与化学农药复配能增加对桃流胶病菌的毒力.

Citrus mandarin (Citrus reticulata) cultivar Shatangju has great economic importance in southern China because of its delicious fruit and medicinal peel. During surveys conducted in 2012 and 2013, stem rot and plant death were observed on 2- to 3-year-old Shatangju grafted seedlings in 21 nurseries located in three regions (Yangjiang City, Sihui City and Deqing County) of Guangdong Province, China. The aims of this study were to characterize the symptoms of seedling stem rot and death; evaluate the incidence and loss; identify the causal agent and evaluate its pathogenicity. The disease was present in all surveyed nurseries but incidence varied with location. A Pythium-like oomycete was consistently obtained from symptomatic plants collected in the nurseries surveyed. On the basis of morphological and cultural characteristics and combined phylogenetic analysis of three DNA sequences including the internal transcribed spacer region (ITS), mitochondrial CoxI and CoxII genes, 21 of the isolates obtained were identified as belonging to the species Phytopythium helicoides. The isolates were pathogenic to the scions but not rootstocks of Shatangju mandarin seedlings cultivated in a controlled greenhouse and symptoms identical to that observed in the field were reproduced. The pathogen was re-isolated consistently from the inoculated plants, confirming Koch’s postulates. Seedling stem rot and eventual death caused by P. helicoides represents a serious threat to Shatangju mandarin nursery productivity and longevity in southern China.

研究了地衣芽孢杆菌W10对烟草的促生作用及对生长相关基因表达的影响，结果表明：W10能明显增加烟草根长、株高、鲜质量；与对照相比，W10处理烟草后茉莉酸信号通路标志基因COI1和调节植物细胞生长的扩展基因NtEXP2、NtEXP6都不同程度上调表达。

Objective] To screen the effective biopesticides and their mixtures with fungicides against the pathogen Phomopsis amygdali of peach shoot blight.[ Method] The virulence of 10 biopesticides against P.amygdali was tested in the laboratory by mycelial growth inhibition method, and then the mixtures of biopesticides with high virulence and prochloraz, carbendazim and diniconazole respectively, were detected for their laboratory toxicities.[Result] Zhongshengmycin had the strongest virulence, with EC50 value of 0.041 9 μg/mL, and shenqinmycin and tetramycin had better antimicrobial effects, with EC50 values of 0.231 4 and 0.655 4 μg/mL, respectively.When shenqinmycin was mixed with the three fungicides in different proportion, the synergistic ratio (SR) values were all greater than 1.5, having increased toxicity effect.When mixed with the three fungicides, zhongshengmycin and tetramycin had mixed ratios for additive and synergistic toxicity effects. [ Conclusion] The study indicates that the biopesticides mixed with chemical fungicides can increase the toxicity to P.amygdali.

Peptides and small molecules produced by both the plant pathogen Phytophthora and host plants in the apoplastic space mediate the relationship between the interplaying organisms. Various Phytophthora apoplastic effectors, including small cysteine-rich (SCR) secretory proteins, have been identified, but their roles during interaction remain to be determined. Here, we identified an SCR effector encoded by scr96, one of three novel genes encoding SCR proteins in P. cactorum with similarity to the P. cactorum phytotoxic protein PcF. Together with the other two genes, scr96 was transcriptionally induced throughout the developmental and infection stages of the pathogen. These genes triggered plant cell death (PCD) in the Solanaceae, including Nicotiana benthamiana and tomato. The scr96 gene did not show single nucleotide polymorphisms in a collection of P. cactorum isolates from different countries and host plants, suggesting that its role is essential and non-redundant during infection. Homologues of SCR96 were identified only in oomycetes, but not in fungi and other organisms. A stable protoplast transformation protocol was adapted for P. cactorum using green fluorescent protein as a marker. The silencing of scr96 in P. cactorum caused gene-silenced transformants to lose their pathogenicity on host plants and these transformants were significantly more sensitive to oxidative stress. Transient expression of scr96 partially recovered the virulence of gene-silenced transformants on plants. Overall, our results indicate that the P. cactorum scr96 gene encodes an important virulence factor that not only causes PCD in host plants, but is also important for pathogenicity and oxidative stress tolerance.

为明确地衣芽孢杆菌(Bacillus licheni ormis)W10菌株的生防效果,采用菌丝生长、产孢、分生孢子萌发和芽管伸长抑制及室内接种方法,研究W10菌液、无菌滤液和抗菌蛋白对桃褐腐病菌(Moniliniafructicola)的抑制作用.结果表明:W10菌液、无菌滤液和抗菌蛋白均能明显抑制病菌菌丝生长、产孢、孢子萌发和芽管伸长.稀释5倍的无菌滤液和1.333 mg·mL-1抗菌蛋白对病菌生长的抑制率分别为85.2％和100％;稀释2倍的无菌滤液和6.666mg·mL-1抗菌蛋白的产孢抑制率分别为84.0％和100％;1×109 cfu·mL-1菌液和1.333 mg·mL-1抗菌蛋白能完全抑制孢子萌发和芽管伸长,稀释2倍的无菌滤液的抑制率也在80％以上.此外,还能显著破坏菌丝形态,使菌丝畸形、原生质渗漏、细胞壁破损和菌丝断裂.离体果实接种试验表明,W10菌液和抗菌蛋白能明显推迟桃果发病,降低果腐率,抑制病斑扩展.因此,地衣芽孢杆菌W10是桃褐腐病潜在的生防因子.

The biocontrol effects of Bacillus licheniformis W10 bacterial suspension and its antifungal protein on peach brown rot caused by Monilinia fructicola in storage peach fruits and the effects on fruit quality were investigated. The results showed that the fruit disease suppression of B. licheniformis W10 bacterial suspension and antifungal protein were significantly higher than that of the control. Inoculation of bacterial suspension and antifungal protein prior to M. fructicola gave a better biocontrol effect, and the higher concentrations of bacterial (1 × 1010 cfu · mL−1) and antifungal protein (3.0 mg · mL−1) performed better control effects. The environmental conditions, such as temperature and humidity, affected biocontrol effects of W10 bacterial suspension and antifungal protein. The influence of environment conditions on the activity of antifungal protein was less than that on bacterial suspension. Moreover, lower temperature (4 °C) and relative humidity (RH 70%–75%) were favorable to prevent peach brown rot by W10 bacterial suspension and its antifungal protein. The W10 bacterial suspension and antifungal protein amended with calcium [0.1% Ca(NO3)2] could enhance the biocontrol effects, and obviously put off the occurrence of peach brown rot. In addition, the bacterial suspension and antifungal protein significantly reduced the natural decay rates of peach fruits during storage, and the effects were equal to carbendazim. Moreover, both W10 bacterial suspension and antifungal protein treatments did not have effects on external and internal fruit appearance, such as chromatic aberration parameter L* of flesh, flesh firmness, soluble solids content and weight loss. Therefore, the B. licheniformis W10 is a potential biocontrol factor for peach brown rot.

在培养基单因子筛选试验基础上,采用响应曲面法对地衣芽孢杆菌W10菌株发酵培养基组成进行优化.结果表明:最佳发酵培养基配方(g·L-1)为黄豆饼粉10.13、玉米淀粉16.89、蛋白胨1.13、葡萄糖2.41、硫酸铵7.96、硫酸镁0.45、磷酸氢二钾0.45、氯化钠4.50.用优化培养基对W10菌株进行发酵培养,发酵液对灰葡萄孢的抑菌率为90.37％,比优化前抑菌率(73.90％)提高22.3％.

[Objective] The aim of this paper was to understand chemical control time against shoot blight of peach. [Method]Control efficacy of spraying fungicides on peach shoot blight at two different time before flowering period was tested in field trials. [Result] The spraying 45%Prochloraz EW( 900-fold dilution) and 50% Carbendazim WP( 500-fold dilution) on March 4,and 10% Difenoconazole WDG( 500-fold dilution) and 50% Carbendazim WP( 500-fold dilution) on March 19 was the best against diseased twigs,with the control effects of 73. 8% and63. 2% for peach varietyHujingmiluandLiutiaobaifeng20 days after the spray two times,respectively,and with the effects of 68. 3% and62. 9% 50 days after the spray. Furthermore,the control effects of fungicides only on March 4 and March 19 were 23. 8%-39. 7% 50 days after the spray. [Conclusion]The study indicated that successive spraying of fungicides two times in early spring before flowering period has significant control effects against peach shoot blight in field.