植物抗软腐病基因工程的策略及研究进展

通过基因工程的手段提高植物对软腐病的抗性已被认为是一条切实可行的途径。目前已有大量研究证实,利用植物与病原菌互作的相关基因、病原菌致病性的相关基因以及杀菌肽基因可以或有望提高植物对软腐病的抗性。文章对以上几个方面及相关的抗软腐病基因工程的一些策略与研究进展进行了论述。     

贡柑黑腐病病原菌的分子鉴定

贡柑黑腐病是近年来在贡柑上发生的一种新型病害,该病害可导致嫩梢快速枯死,大量嫩叶脱落,严重影响树势和产量。本研究从病区采集发病枝叶,对贡柑黑腐病病原菌进行分离、纯化,并采用rDNA-ITS序列分析技术对其进行分子鉴定。结果表明,贡柑黑腐病病原菌为链格孢属（Alternaria Nees）真菌,rDNA-ITS序列与GenBank中芸薹链格孢菌(Alternaria brassicae (Berk.) Sacc.)和柑桔链格孢菌(Alternaria citri Ellis & Everhart)的ITS序列的同源性为99%。结合形态学特征,推断该病原菌为柑桔链格孢菌。     

10种杀细菌剂对火龙果细菌性茎腐病菌的毒力测定

采用抑菌圈法,对10种杀细菌剂在火龙果茎腐病病原上进行了毒力测定。结果表明：72%农用硫酸链霉素可溶粉剂、53.8%氢氧化铜水分散颗粒剂（杜邦TM）、45%代森铵水剂、47%春雷?王铜可湿性粉剂、50%福美双可湿性粉剂和3%中生菌素可湿性粉剂对火龙果茎腐病病原均有一定抑制效果。其中,45%代森铵水剂和50%福美双可湿性粉剂防治效果较好,抑制率分别达到78.88%和71.42%,其直线回归方程分别为y=2.380 2.477x,y=2.896 1.146x,EC50分别为0.4929,0.0240。72%农用硫酸链霉素可溶粉剂效果次之,抑制率达到66.67%,直线回归方程为y=1.020 1.040x,EC50值为2.5686。这3种药剂可作为火龙果茎腐病田间药效试验的首选药剂。     

江西永修凤凰山桃实腐病病原鉴定

桃实腐病是江西永修凤凰山桃园发生的一种新的重要病害,其主要危害挂果期的果实,严重影响桃产量和品质。病果主要表现为脐部变褐腐烂,并在病部表面密生黑色小点（分生孢子器）,病果易脱落。本文对病果进行了组织分离,对获得的病菌纯培养进行了回接,同时对病菌的形态及大小观察和测量,结果表明引起凤凰山桃实腐病的病原菌为核果果腐拟茎点菌（Phomopsis amygdalina Canonaco）。     

一株马铃薯干腐病生防菌的鉴定及其抑菌活性研究

镰孢菌引起的马铃薯干腐病是影响马铃薯贮存期间产量与品质的重要病害之一。寻找高效低毒的防治马铃薯干腐病的生防药剂对提高马铃薯的产量与质量具有重要意义。本研究以腐皮镰孢Fusarium solani为病原菌, 测定分离自马铃薯块茎的菌株2-1-9-CJK-2的抑菌活性。该菌株发酵液、无菌发酵液、菌悬液、胞内组分以及挥发性有机物对腐皮镰孢均具有抑菌活性, 抑制率分别为93.37%、61.92%、80.82%、44.01%和49.16%。结合形态学、生理生化特征和多基因系统发育树构建鉴定其为贝莱斯芽胞杆菌Bacillus velezensis。马铃薯切片试验和马铃薯块茎试验表明, 当菌株发酵液浓度为1×109 cfu/mL时, 对病原菌的抑制效果最好。除此之外, 菌株可以分泌蛋白酶、淀粉酶、纤维素酶, 具有溶磷能力和生物膜形成能力, 但不具有溶血性, 并且对供试的其他6种病原真菌有较强的抑菌活性。研究表明贝莱斯芽胞杆菌2-1-9-CJK-2抑菌谱广, 具有生物安全性, 对腐皮镰孢有较强的抑菌活性, 在植物病害防治中具有一定的应用潜力, 可为马铃薯干腐病生防制剂的开发奠定基础。     

A novel glycoside hydrolase 74 xyloglucanase CvGH74A is a virulence factor in Coniella vitis

Grape white rot is a destructive fungal disease occurring worldwide. Recently, Coniella vitis was identified as the predominant pathogen causing this disease in China. As the periderms of grape shoots are severely degraded by C. vitis, it was speculated that cell wall-degrading enzymes (CWDEs) might play a key role in the pathogenesis of this disease. Therefore, this study aimed to examine the hydrolytic activity of the CWDEs of C. vitis. The results showed that xylanase (Xy) and xyloglucanase (XEG) had high levels of hydrolytic activity both in vitro and in vivo. Furthermore, a high-virulence fungal strain exhibited higher levels of Xy and XEG activities compared with a low-virulence strain. The genome of the fungus was found to harbor two XEG-coding genes CvGH74A and CvGH74B, which belonged to the glycoside hydrolase (GH)74 family. The expression level of CvGH74A was found to be high during pathogen infection. CvGH74A gene deletion mutants were generated using the split-marker method. The deletion of CvGH74A decreased both the hydrolytic activities of XEG and Xy and also the ability of the fungus to infect the grape leaves. No differences in the hyphal growth, morphology of colonies, or conidiation were found between the ΔCvGH74A mutant strains and the wild-type strain. Together, these results suggested that CvGH74A acted as an important virulence factor, and its enzymatic activity might regulate the virulence of the pathogen. This study was novel in reporting that GH74 XEG acted as a virulence factor in C. vitis.     

黄瓜绵腐病病原菌鉴定及防治药剂的筛选

为有效防控黄瓜绵腐病,本研究对自江西省南昌市南昌县武阳镇和八一乡采集的黄瓜绵腐病病样进行分离,基于致病性测定、形态学特征和分子生物学方法对其进行鉴定,并采用菌丝生长速率法测定甲霜灵、霜霉威、霜脲氰、烯酰吗啉4种常用杀菌剂对该病原菌和辣椒疫霉Phytophthora capsici的抑制效果。结果表明,自武阳镇和八一乡采集的病样经分离、纯化培养后分别获得12株和10株菌株;自武阳镇分离的病原菌的藏卵器柄较直,藏卵器大小为18.1~32.9 μm;自八一乡分离的病原菌的藏卵器柄较弯曲,藏卵器大小为15.1~25.3 μm;结合致病性测定及rDNA-ITS测序结果,将自武阳镇分离的病原菌鉴定为瓜果腐霉Pythium aphanidermatum,将自八一乡分离的病原菌鉴定为德里腐霉Py.deliense。室内毒力测定结果表明,甲霜灵对瓜果腐霉和德里腐霉抑制效果最好,EC₅₀分别为0.71 μg/mL和0.25 μg/mL;霜脲氰次之,EC₅₀分别为3.68 μg/mL和3.36 μg/mL,甲霜灵、霜霉威、霜脲氰3种杀菌剂对瓜果腐霉、德里腐霉和辣椒疫霉的抑制效果差别不大,但烯酰吗啉对瓜果腐霉、德里腐霉和辣椒疫霉抑制效果差异明显,EC₅₀分别为18.06、33.58和0.18 μg/mL,表明防治黄瓜绵腐病优先选择甲霜灵。     

Strategy to identify resistance to white mould associated with high yield for irrigated common bean in Brazil

Development of common bean cultivars with partial white mould resistance through breeding techniques has been a challenge in Brazil. As yet, lines/cultivars from breeding programmes have not been investigated for resistance; therefore, this study screened 107 lines/cultivars for their reactions to white mould in 14 preliminary trials conducted under irrigation. Thirteen resistant lines/cultivars (three of Andean origin) and six Mesoamerican cultivars (three intermediately resistant and three susceptible) were selected for further investigation. These lines/cultivars and the resistant control A195 were evaluated in six advanced trials and two straw tests to assess the effectiveness of the screening procedure. In 11 preliminary trials, screenings were performed under moderate/high or higher disease pressure. These pressures occurred in two advanced trials in which, when yields were averaged across moderate/high and high pressures, 10 Mesoamerican lines/cultivars selected for resistance yielded 14%, 23%, and 38% more than intermediately resistant cultivars, A195, and susceptible cultivars, with median disease ratings (1–9 scale) of 4.5, 5.7, 5.7, and 6.7, respectively. In the straw test, three Andean lines/cultivars (A195 included) and two susceptible cultivars in the field were among those with the highest levels of physiological resistance. Thus, field rating under high disease pressure and greenhouse rating did not correlate significantly, suggesting that field trials are critical to evaluating resistance and to identifying high-yielding beans. Therefore, lines/cultivars from breeding programmes assessed in field trials may provide a low cost and fast way to identify high-yielding bean cultivars with partial resistance to white mould in the subtropical southern hemisphere.     

解淀粉芽胞杆菌菌株HRH317对感染串珠镰孢菌玉米幼苗伏马毒素B1含量及相关防御酶活性的影响

为探讨解淀粉芽胞杆菌Bacillus amyloliquefaciens菌株HRH317对感染串珠镰孢菌Fusarium moniliforme玉米幼苗产生伏马毒素B_1(FB_1)的影响,采用牛津杯法测定菌株HRH317对串珠镰孢菌的抑制活性,并通过浸种处理进行盆栽试验,应用高效液相色谱技术对生长至3叶期后不同时间玉米幼苗叶片中FB_1含量进行测定,同时于室内测定玉米幼苗叶片防御酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)的活性。结果表明:解淀粉芽胞杆菌菌株HRH317能明显抑制串珠镰孢菌生长,抑菌圈直径平均可达33.31 mm;玉米幼苗生长至3叶期后1～6 d,菌株HRH317能有效抑制玉米植株体内FB_1含量,经串珠镰孢菌分生孢子悬浮液与菌株HRH317菌悬液1∶1混合液处理玉米种子后,对幼苗中FB_1的抑制率为59.20%～75.70%;而玉米种子先接种菌株HRH317菌悬液后接种串珠镰孢菌分生孢子悬浮液处理对幼苗中FB_1的抑制率为76.77%～88.10%。且这2种处理中幼苗叶片的SOD、CAT、PAL和POD活性均较对照有不同程度提高,其峰值是对照的1.24～5.45倍。表明解淀粉芽胞杆菌菌株HRH317可通过抑制FB_1产生来降低串珠镰孢菌对玉米幼苗的侵害,同时能诱导玉米植株体内防御酶活性的表达而增强其系统抗性,在防治玉米穗腐病方面具有潜在的应用价值。     

菌剂与钾肥配施对连作魔芋生长、生理代谢和发病率的影响

以安康当地花魔芋为材料进行盆栽试验,研究娄彻氏链霉菌（Streptomyces rochei）菌剂D74与钾肥配施对连作魔芋生物学性状、块茎产量、叶绿素含量、活性氧代谢、渗透调节物质含量及软腐病的影响,为魔芋连作障碍微生物-化学双重修复提供理论依据。结果表明：菌剂与钾肥配施对连作魔芋具有一定的促生防病作用,使魔芋单株块茎鲜质量和生长系数分别较对照显著增加86.7%和85.9%,软腐病病情指数降低 33.0%。配施处理中,钾肥和菌剂分别在促进魔芋块茎膨大和抑制软腐病方面发挥主导作用。菌剂与钾肥配施促进魔芋叶片叶绿素的合成,其叶绿素a及叶绿素(a+b)含量分别较对照显著增加51.0%及34.6%,同时叶绿素a/b及叶绿素(a+b)/类胡萝卜素比值分别提高41.8%及22.2%。配施的菌剂效应对叶绿素合成的促进作用占主导地位。菌剂与钾肥配施显著提高魔芋的诱导抗性,其叶片过氧化物酶活性、可溶性蛋白和可溶性糖含量分别为对照的3.1倍、2.9倍和2.2倍。采用主成分分析法对魔芋相关19个生理生化指标分析得出特征值、贡献率、累积贡献率和主成分载荷矩阵,提取3个主成分（叶绿素b、叶幅和相对防效）综合评价菌剂与钾肥配施对魔芋连作障碍的修复效果。不同施肥处理综合得分高低顺序为：菌剂与钾肥配施>单施钾肥>单施菌剂>不施肥对照。综上所述,娄彻氏链霉菌菌剂D74与钾肥配施能提高连作魔芋产量并降低植株发病率,有效缓解连作障碍。