Biological Control to Protect Watermelon Blossoms and Seed from Infection by Acidovorax avenae subsp. citrulli

ABSTRACT The efficacy of biological control seed treatments with Pseudomonas fluorescens (A506), Acidovorax avenae subsp. avenae (AAA 99-2), and an unidentified gram-positive bacterium recovered from watermelon seed (WS-1) was evaluated for the management of bacterial fruit blotch (BFB) of watermelon. In growth chamber and greenhouse experiments, seed treated with AAA 99-2 displayed superior disease suppression, reducing BFB transmission by 96.5%. AAA 99-2, P. fluorescens A506, and Kocide also suppressed the epiphytic growth of A. avenae subsp. citrulli when applied to attached watermelon blossoms 5 h prior to inoculation. Watermelon blossom protection reduced seed infestation by A. avenae subsp. citrulli. From blossoms treated with 0.1 M phosphate buffered saline (PBS), 63% of the resulting seed lots were infested with A. avenae subsp. citrulli. In contrast, for blossoms protected with WS-1, Kocide, P. fluorescens A506, and AAA 99-2, the proportion of infested seed lots were 48.3, 21.1, 24.1, and 13.8%, respectively. The effect of blossom treatments on seed lot infestation was statistically significant (P = 0.001) but WS-1 was not significantly different from PBS. These findings suggest that blossom protection with biological control agents could be a feasible option for managing BFB.     

Bio-PCR方法检测葫芦科作物种子携带 西瓜嗜酸菌的特异性引物筛选

 由西瓜嗜酸菌(Acidovorax citrulli)引起的细菌性果斑病是一种毁灭性的种传病害,可为害多种葫芦科作物并造成重大经济损失。该病原菌为检疫性有害生物,种子带菌是田间病害发生的最重要初侵染来源,因此,种子健康检测成为病害综合防控过程中的重要环节。Bio-PCR是当前种子携带细菌检测的常用方法,而特异性引物的选择和使用是检测的关键。本研究使用已报道的7对引物对17株西瓜嗜酸菌、10株嗜酸菌属其它种的菌株和6株其它属的植物病原细菌进行了Bio-PCR检测,筛选出对西瓜嗜酸菌特异性最好的引物为SEQID4^m/SEQID5。研究表明:使用该引物对西瓜嗜酸菌MH21纯菌菌悬液的检测限度为10² CFU·mL^-1;在人工添加菌悬液的模拟带菌西瓜种子中,使用ASCM和EBBA两种半选择性培养基结合引物SEQID4^m/SEQID5进行Bio-PCR检测,ASCM对种子中带菌量的检测限度可达到0.01 CFU·g^-1,EBBA对种子中带菌量的检测限度为0.1 CFU·g^-1。     

Role of Blossoms in Watermelon Seed Infestation by Acidovorax avenae subsp. citrulli

ABSTRACT The role of watermelon blossom inoculation in seed infestation by Acidovorax avenae subsp. citrulli was investigated. Approximately 98% (84/87) of fruit developed from blossoms inoculated with 1 x 10(7) or 1 x 10(9) CFU of A. avenae subsp. citrulli per blossom were asymptomatic. Using immunomagnetic separation and the polymerase chain reaction, A. avenae subsp. citrulli was detected in 44% of the seed lots assayed, despite the lack of fruit symptoms. Furthermore, viable colonies were recovered from 31% of the seed lots. Of these lots, 27% also yielded seedlings expressing bacterial fruit blotch symptoms when planted under conditions of 30 degrees C and 90% relative humidity. A. avenae subsp. citrulli was detected and recovered from the pulp of 33 and 19%, respectively, of symptomless fruit whose blossoms were inoculated with A. avenae subsp. citrulli. The ability to penetrate watermelon flowers was not unique to A. avenae subsp. citrulli, because blossoms inoculated with Pantoea ananatis also resulted in infested seed and pulp. The data indicate that watermelon blossoms are a potential site of ingress for fruit and seed infestation by A. avenae subsp. citrulli.     

应用免疫金探针定位和定量测定稻种中的水稻细菌性条斑病菌

本文首次报道用免疫金染色和免疫金银染色对水稻细菌性条斑病病种的带菌部位及带菌量的研究。结果表明:病原细菌分布于谷壳、籽实皮、胚和胚乳,主要集中在谷壳上。谷壳外表带菌量多于内表面。籽实皮外表面由于受谷壳污染较之内表面的带菌量多。籽实皮内表面、胚和胚乳的带菌量均较少,差异不显著。谷壳、谷壳内表面、籽实皮、胚乳(或胚)的带菌量之比为36:13:9:1。     

应用免疫金探针定位和定量测定稻种中的水稻细菌性条斑病菌

 本文首次报道用免疫金染色和免疫金银染色对水稻细菌性条斑病病种的带菌部位及带菌量的研究。结果表明:病原细菌分布于谷壳、籽实皮、胚和胚乳,主要集中在谷壳上。谷壳外表带菌量多于内表面。籽实皮外表面由于受谷壳污染较之内表面的带菌量多。籽实皮内表面、胚和胚乳的带菌量均较少,差异不显著。谷壳、谷壳内表面、籽实皮、胚乳(或胚)的带菌量之比为36:13:9:1。     

西瓜嗜酸菌趋化性的初步研究

瓜类细菌性果斑病是世界范围的检疫性细菌病害,病原菌为西瓜嗜酸菌,带菌种子为主要侵染源。病原细菌在寄主表面的定殖能力与其致病能力关系密切,而趋化性是决定定殖能力的关键因素之一,研究不同物质对西瓜嗜酸菌趋化性的影响对防治瓜类细菌性果斑病具有重要意义。本文采用毛细管法,研究了碳源、氨基酸、有机酸及其他物质对西瓜嗜酸菌趋化性的影响。结果表明,所测碳源中,麦芽糖、葡萄糖、乳糖、蔗糖和半乳糖均显著促进西瓜嗜酸菌的趋化性;所测氨基酸中,L-精氨酸、L-天冬氨酸、L-组氨酸、L-谷氨酸、L-亮氨酸、L-缬氨酸、L-谷氨酰胺和L-丙氨酸显著促进西瓜嗜酸菌的趋化性;所测有机酸中,琥珀酸、半乳糖醛酸和酒石酸显著促进西瓜嗜酸菌的趋化性;氯化钠、硫酸镁等对西瓜嗜酸菌的趋化性无显著影响。     

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds was investigated. Citron melon seed lots infected with A. citrulli were generated in the field by inoculating either the pistils (stigma) or pericarps (ovary wall) of the female blossoms. Seventeen A. citrulli isolates from 14 different haplotypes belonging to two different groups (group I and II) were used for inoculation. After confirming that 100% of seed lots were infected, they were stored at 4°C and 50% RH for 7 years. After storage, the viability of A. citrulli cells from individual lots was determined by plating macerated seeds on semiselective medium as well as growing seeds for 14 days and scoring for bacterial fruit blotch symptoms. The type of A. citrulli isolate (group I or group II) used did not significantly influence bacterial survival. However, A. citrulli survival was significantly greater in seed lots generated via pistil inoculation (52·9 and 29·4%) than via pericarp inoculation (23·5 and 17·6%). Repetitive extragenic palindrome (rep)‐PCR on A. citrulli isolated from citron melon seed lots after storage displayed similar fingerprinting patterns to those of the reference strains originally used for blossom inoculation, indicating that cross‐contamination did not occur. The results indicate that A. citrulli may survive/overwinter in citron melon seeds for at least 7 years and bacterial survival in seed was influenced more by method of blossom inoculation than by the type of bacterial isolate.     

油菜病毒病种子带病毒的初步研究

 感染芜菁花叶型病毒和烟草花叶型病毒的油菜病株种子的种皮内带有病毒,种胚自未成熟到成熟始终不带病毒。
烟草花叶型病毒在种皮内的存活期限长于芜菁花叶型病毒。烟草花叶型毒株61-16号,在"胜利"油菜病株种子的种皮内可以存活26个月以上;芜菁花叶型病毒存活期最长的一个毒株不超过25个月,最短的不到4个月。     

进境西瓜种子中西瓜细菌性果斑病菌的检测鉴定

由嗜酸菌属西瓜种(Acidovorax citrulli)引起的西瓜细菌性果斑病是西瓜等葫芦科作物上的一种极具毁灭性的病害,该病原细菌可以由种子携带传播。本研究通过直接研磨种子,用双抗体夹心酶联免疫吸附测定法(DAS-ELISA)和免疫捕捉聚合酶链式反应法(IC-PCR)对进境的西瓜种子进行检测,并且对PCR产物进一步克隆测序。结果表明,在DAS-ELISA产生阳性结果的样品中,IC-PCR结果也产生阳性。序列分析表明,该序列与已知的该病菌16S rRNA基因的相应序列具有100%同源性。因此,2006年这批来自台湾的西瓜种子携带有嗜酸菌属西瓜种(Acidovorax citrulli)。     

Influence of the amount of blackgram mottle virus in different tissues on transmission through the seeds of Vigna mungo

Blackgram mottle virus (BMoV) was transmitted via up to 16% of seeds of different cultivars of blackgram, as determined by seedling symptom tests. The percentage of seed infection by BMoV as determined by EL1SA was even higher. Seed transmission was highest in cv. PLU-277 (15.9%), followed by cvs T-9 (11.8%), PLU-213 (7.0%) and UH-81-7 (1.3%). Seed transmission was correlated with the amount of virus present in the embryonic axis and later in primary leaves. The presence of virus in the testa alone did not result in its transmission through seeds. Virus concentration in different tissues varied; the mean amount of virus in the three cultivars was found to be 48–1234 ng per embryonic axis, 15–24 ng per cotyledon, and 12–20 ng per testa. The infection of primary leaves through the seed also resulted in systemic infection if the amount of virus in primary leaves exceeded 100 ng/100 mg of tissue. Close agreement was found between the percentage of seedlings with systemic infection and the percentage of seeds and embryonic axes containing more than 100 ng virus. The cultivars that resisted seed transmission contained relatively small amounts of the virus in embryonic axes.     

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.     

应用微滴数字PCR同时检测瓜类种子携带果斑病菌和角斑病菌

细菌性果斑病和角斑病是葫芦科作物两大重要细菌病害,病原菌分别为西瓜嗜酸菌Acidovorax citrulli和丁香假单胞菌黄瓜致病变种Pseudomonas syringae pv.lachrymans。两种菌均可通过种子、种苗带菌进行远距离传播。种子检测是预防和控制这两种病害发生的首要环节。本研究应用微滴数字PCR技术(droplet digital PCR,ddPCR)建立了同时检测种子携带西瓜嗜酸菌和丁香假单胞菌的方法。结果显示:两种细菌菌悬液和DNA样品等浓度混合时,ddPCR能同时检测到两种靶标菌的最低混合菌悬液浓度和最低DNA浓度分别为10³ cfu/mL和10^-3 ng/μL,其检测灵敏度是平行测试的real-time PCR方法的10倍;对于非等浓度混合的菌悬液和DNA样品,两种靶标菌菌悬液按浓度比1∶1000(10³∶10⁶ cfu/mL)混合或其DNA浓度比为1∶10000(2.28×10^-3 ng/μL∶22.8 ng/μL)条件下,ddPCR可检测到低浓度的靶标菌,检测灵敏度同样是real-time PCR的10倍。此外,在人工接菌种子测试中,西瓜、甜瓜单粒种子平均带菌量10⁵~10⁶ cfu/粒时,ddPCR方法可检测到带菌率0.2%(n=500)的西瓜、甜瓜种子样品。将分别携带两种菌的种子按比例1∶10混合时ddPCR方法可以准确检出浓度相对低的靶标菌;而使用相同检测引物的real-time PCR检测方法则只能检出西瓜嗜酸菌和丁香假单胞菌带菌率分别为0.2%和2%(n=500)的甜瓜种子混合样品中的西瓜嗜酸菌,未能稳定检出丁香假单胞菌。综上所述,本研究基于ddPCR技术建立了可同时检测两种重要葫芦科种传细菌的方法,检测结果稳定可靠,丰富了当前种传病原细菌的检测技术体系。     

Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis ( Cmm ) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D₂ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation ( R ² = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g⁻¹ caused disease in 78 plants out of 2000.     

种子引发处理对无籽西瓜幼苗生长的影响和对细菌性果斑病菌消毒的效果

 种子出苗率低和传带细菌性果斑病菌是制约三倍体无籽西瓜生产的主要原因。本试验利用KMnO₄、CuSO₄和ZnSO₄的不同浓度溶液对无籽西瓜种子进行不同时间引发处理, 通过滤纸发芽试验, 观测其对种子发芽和幼苗生长的促进作用;借助平板法测定引发溶液对细菌性果斑病菌FC247的抑制作用, 免疫凝聚试纸条和传统PCR检测引发处理对种子人工接菌FC247的消毒效果。结果表明:0.1% CuSO₄溶液引发处理4 h和0.2% ZnSO₄溶液引发处理24 h, 分别比未引发处理的无籽西瓜种子发芽率提高71.1%和73.3%, 并能显著提高发芽整齐度和幼苗素质;同时, 引发溶液对细菌性果斑病菌有显著抑制作用, 并对人工接菌种子表现出-定程度的消毒效果。     

不适温度条件诱导西瓜嗜酸菌进入VBNC状态的研究

由西瓜嗜酸菌(Acidovoraxcitrulli)引起的瓜类果斑病,是危害西瓜和甜瓜等葫芦科作物的一种典型的种传细菌性病害.已有文章报道西瓜嗜酸菌在铜离子诱导下可进入"有活力但不可培养"(viable but non-culturable,VBNC)状态,并在适当条件下复苏,成为生产中潜在的病害初侵染来源.本文结合实...     

Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments

Rates of seed transmission of Melon necrotic spot virus (MNSV) were estimated in seedlings grown from commercial melon ( Cucumis melo ) cv. Galia F₁ seeds. Seedlings at the cotyledon stage and adult plants were assayed for MNSV by DAS-ELISA and RT-PCR. None of the seedling groups tested positive for MNSV by ELISA. The proportion of seedlings infected with MNSV was at least 7 and 8% in seed lots 05 and 06, respectively, as estimated from RT-PCR analysis of grouped seedlings. Fourteen and eight grouped samples (10 seedlings per group), of a total of 200 and 100 seedlings, respectively, grown from infected seeds were MNSV-positive in seed lots 05 and 06, respectively, corresponding to seed-to-seedling transmission rates of 11·3 and 14·8%, respectively. Several seed-disinfection treatments were evaluated for their ability to prevent seed transmission of MNSV. The results suggest that a treatment of 144 h at 70°C can be used to eradicate MNSV in melon seeds without hindering germination.     

A method for detection of seedborne bacterial diseases in tomato seeds

A method for detection and quantitative estimation of tomato seedborne pathogenic bacteria has been developed. It enables detection in a 7 g tomato seed sample of as few as ten colony-forming units per gram tomato seeds of the following seedborne pathogens of tomato:Pseudomonas syringae pv. tomato,Pseudomonas corrugata, Xanthomonas campestris pv.vesicatoria, andClavibacter michiganense subsp.michiganense. With representative seed samples, the method employs dry grinding, weighing, bacterial extraction and quantitative calculation on selective or semi-selective medium. The efficiency of this method was tested by diluting pathogen-free seed lots with naturally or artificially infested tomato seeds. This procedure enables one to determine the minimal threshold of pathogen which can be detected by this method on media, in comparison with the percentage of diseased seedlings developed from the same seed lots in the growth chamber or in the greenhouse.     

<Emphasis Type="Italic">Indian citrus ringspot virus:</Emphasis> localization of virus in seed tissues and evidence for lack of seed transmission

Indian citrus ringspot disease is an important viral disease in kinnow mandarin orchards where disease incidence up to 100% has been recorded. The disease is caused by Indian citrus ringspot virus (ICRSV), a positive sense flexuous RNA virus. The transmission of ICRSV is generally through budwood. Association of ICRSV with pollens of naturally infected flowers from cv. ‘Kinnow’ mandarins has been shown previously and this study demonstrates the presence of ICRSV in seed tissues. DAC-ELISA revealed the presence of virus in seed coats but not in embryo and endosperm of seeds collected from the fruits of ICRSV-infected Kinnow plants. Of the infected seed coats, 18% were found to harbor the virus. The seedlings in the grow-out test did not show any symptom for 2 years and the virus could not be detected in seedlings by DAC-ELISA and RT-PCR. The present study indicated that ICRSV could be localized in the testa of seeds but its transmission to progeny was not observed.     

The roles of light and pericarp on seed dormancy and germination in feral Raphanus sativus (Brassicaceae)

The timing of seed germination may determine the success of a weed species in an agroecosystem, and its expression is modulated by environmental conditions, but also by seed physiology and anatomy. The aims of this study were to investigate the roles of light, pericarp, dry storage and cold stratification on seed dormancy and germination in feral radish, a troublesome agricultural weed in temperate zones of the Americas that reduces crop yields. To this end, we used isolated intact pods and extracted seeds to test germination over time under contrasting temperature, light and storage conditions. Here, we showed that fresh seeds were non‐dormant, but that light and the presence of the pericarp reduced germination, especially under low temperatures. The pericarp reduced the final water content absorbed by seeds inside pods and decreased absorption/dehydration rates. The pericarp showed several small lignified cell layers in the endocarp, and x‐ray images displayed the lack of space between the partially embedded seed and the endocarp. Dry storage and cold stratification were ineffective in breaking the dormancy imposed by the pericarp. The apparent requirement for darkness and the mechanical restriction of the pericarp may have the potential to induce dormancy, spreading the timing of seed germination over a more extended period and hindering the control of feral radish.     

Pathways of bacterial invasion and watermelon seed infection by Acidovorax citrulli

This study explored the pathways of ingress of Acidovorax citrulli, the causal agent of bacterial fruit blotch of cucurbits, into watermelon seeds. Up until 7 days post‐inoculation (DPI), a significantly higher percentage of watermelon seeds was infected with A. citrulli when the bacteria were applied (c. 1 × 10⁶ colony‐forming units) to stigmas versus ovary pericarps of female flowers. Immunofluorescence microscopy showed that, with stigma inoculation, A. citrulli colonized style and ovary tissues by 1 DPI, and the bacteria co‐localized with pollen germ tubes in these tissues. With ovary pericarp inoculation, A. citrulli cells penetrated the epicarp and mesocarp tissues by 1 DPI but did not reach endocarp until 4 DPI. Finally, manual pollination followed by stigma inoculation led to >53% A. citrulli‐infected seed lots, while A. citrulli was not detected in seeds/ovules generated by stigma inoculation without pollination (chemically induced parthenocarpy). These results show that stigma inoculation results in faster colonization of watermelon ovules by A. citrulli than pericarp inoculation, even though there is no difference in the levels of infection in mature seeds. The data also indicate that pollen germ tubes play an important role in A. citrulli ingress into watermelon seeds via stigmas.