烟草曲叶病毒可通过烟粉虱侵染番木瓜引致曲叶病

 番木瓜(Carica papaya L)在我国的广东、广西、福建和海南等省区广泛种植。60年代以来,番木瓜环斑病毒病(Papaya Ringspot Virus,PRV)逐年加剧,现几乎所有的番水瓜产区均有发生,并被认为是我国番木瓜上发现的唯一的一种病毒病。     

二氢茉莉酸丙酯诱导烟草抗黑胫病作用研究

研究了二氢茉莉酸丙酯(PDJ)对烟草黑胫病的控制作用,结果表明:离体条件下,PDJ对烟草黑胫病Phytophthora parasitica Var. nicotianae的菌丝生长、孢子囊产生、游动孢子释放及萌发均无明显的抑制作用;PDJ处理后显著减轻了烟草幼苗黑胫病的病情,其最佳处理浓度为100 mg/L,最佳处理时间是接种前3 d,持效期在15 d以上;并且PDJ 100 mg/L处理烟苗下部第3片叶后,可使上部未处理叶片病情显著减轻。表明PDJ诱导了烟草幼苗对黑胫病的抗性。     

白背飞虱群体接毒条件的优化及抗病毒药剂对南方水稻黑条矮缩病的防效

为比较氨基寡糖素等抗病毒诱导剂和嘧肽霉素等抗病毒剂对南方水稻黑条矮缩病的防效,从稻苗生育期、饲毒与接毒时间等方面优化白背飞虱群体接毒条件,对不同药剂处理的稻苗进行人工接毒,并分别于接毒前后喷施抗病毒诱导剂和抗病毒剂,比较不同药剂防效的差异。结果显示,3叶1心期稻苗分别饲毒与接毒12 h较适合室内白背飞虱的群体接毒。抗病毒诱导剂中,100 mg/kg氨基寡糖素和60 mg/kg超敏蛋白的效果较好,病情指数分别为54.86和52.86,防效分别为22.17%和24.99%。抗病毒剂中,240 mg/kg嘧肽霉素的效果较好,病情指数为50.95,防效为28.67%;其次为120 mg/kg嘧肽霉素和600 mg/kg毒氟磷,病情指数分别为55.71和59.05,防效为22.00%和17.34%。     

戊唑醇防治小麦赤霉病施药时期及安全性评价

为了明确戊唑醇防治小麦赤霉病的关键施药时期及其安全性,进行了戊唑醇防治小麦赤霉病施用浓度和时期的田间试验。结果表明,25%戊唑醇EC在有效成分150g/hm2和75g/hm2两个施用剂量下对小麦赤霉病的防治效果没有显著差异,对病穗率和病情指数的防治效果分别为83.7%、86.0%和85.1%、88.5%;25%戊唑醇EC在有效成分150g/hm2施用剂量下在齐穗期到灌浆初期喷施对小麦安全,以盛花期施药对小麦赤霉病的防治效果最好,对病穗率和病情指数的防治效果分别为80.6%和84.1%,其次为齐穗期和初花期施药,对病穗率和病情指数的防治效果分别为53.9%、53.3%和54.6%、56.8%,灌浆初期施药的防治效果最差。     

华南番木瓜环斑病毒的鉴定、提纯与性质的初步研究

 为害华南番木瓜的病毒以番木瓜科(Caricaceae)和葫芦科(Cucurlitaceae)中若干植物为寄主,范围狭窄。病毒的稀释终点为10^-2~10^-3;热灭活点为50~55℃;体外存活期为8~16小时。病毒颗粒长约600~800nm、宽约10~15nm的略为弯曲的细丝。在感病番木瓜组织的细胞内,电镜观察到典型的风轮状内含体(pinwheel inclusion body)。病毒具有马铃薯Y病毒组成员的典型特征,可认为是国外报道的番木瓜环斑病毒的中国株系,初步命名为华南番木瓜环斑病毒。     

番木瓜环斑病毒南瓜分离物外壳蛋白基因的克隆及序列分析

利用电镜和酶联免疫法在云南省采集到的5份南瓜病样中检测到番木瓜环斑病毒(Papayaring spot virus,PRSV)。为了进一步从分子水平确定云南省南瓜病毒病原种类,并为下一步转基因育种提供抗性基因,采用反转录PCR(RT-PCR)方法扩增了5个分离物的外壳蛋白(coat protein,CP)基因片段,并克隆到pGEM-T载体中。核苷酸序列测定表明,番木瓜环斑病毒石屏分离物(PRSV-SP)和番木瓜环斑病毒蒙自分离物(PRSV-MZ)的CP基因长873nt,编码290个氨基酸,番木瓜环斑病毒峨山分离物(PRSV-ES)、番木瓜环斑病毒版纳分离物(PRSV-BN)和番木瓜环斑病毒宾川分离物(PRSV-BC),3个分离物CP基因长867nt,编码288个氨基酸。PRSV5个分离物核苷酸序列的同源性在94%以上,氨基酸序列的同源性在96%以上。与国内外17个分离物相比,核苷酸序列同源性为89.6%~98.7%,氨基酸序列同源性为86.5%~99.6%。其中PRSV-SP和来自于越南分离物PRSV-V47无论是核苷酸序列,还是氨基酸序列同源性都达到了最高,而5个分离物与来自于巴西(PRSV-BR)、美国(PRSV-USA)、墨西哥(PRSV-Y)核苷酸序列同源性均低于90%。     

外源茉莉酸甲酯对紫花苜蓿尖孢镰刀菌根腐病抗病性的作用

 本研究以紫花苜蓿品种、茉莉酸甲酯(Methyl Jasmonnate, MeJA)浓度、MeJA诱导天数为处理因素,按照四因素三水平[L₉(3⁴)]的正交试验设计,通过测定发病情况和相关生理生化指标来探究MeJA对紫花苜蓿尖孢镰刀菌根腐病抗病性的作用。结果表明,施用外源MeJA能够降低甘农3号、龙牧803和中苜一号的发病率和病情指数。外源MeJA诱导处理可增强超氧化物歧化酶和苯丙氨酸解氨酶的活性,但对过氧化物酶的影响相对较小;经诱导处理后地上生物量鲜重和干重也有显著增加,而可溶性蛋白质的含量却显著降低。方差分析和极差分析结果表明,MeJA诱导处理存在品种、诱导浓度和诱导天数间的差异,其中MeJA浓度是影响各测定指标的主要因素。通过因子综合分析得出,本次研究中甘农3号,用MeJA 0.1 mg·mL^-1诱导处理并同时接种对尖孢镰刀菌引起的根腐病的抑制效果最佳。     

茉莉酸甲酯对水稻白叶枯病的诱导抗性及相关防御酶活性的影响

为探索茉莉酸甲酯(methyl jasmonate,MeJA)诱导水稻抗白叶枯病的效应,采用MeJA喷雾处理剪叶接种法,测定MeJA对水稻幼苗的白叶枯病病情指数、白叶枯病病菌Xanthomonas oryzae pv.oryzae的抑菌效果及对叶片过氧化物酶(peroridase,POD)、过氧化氢酶(catalase,CAT)、超氧化物歧化酶(superoxide dismutase,SOD)、多酚氧化酶(polyphenol oxidase,PPO)和苯丙氨酸解氨酶(phenylalnaine ammonialyase,PAL)等相关防御酶活性的影响.0.05 ～ 2.0 mmol/L的MeJA能降低水稻幼苗白叶枯病的病情指数,但对水稻白叶枯病菌无直接抑菌活性;0.1 mmol/L MeJA的诱导效果最好,处理48h后,感病品种温229和抗病品种嘉早312的诱导效果分别为73.18％和70.43％;0.05 ～2.0mmol/L的MeJA处理水稻叶片中POD、CAT、SOD、PPO和PAL活性呈上升趋势.研究表明MeJA能诱导水稻幼苗对白叶枯病的抗性,且诱导抗性的产生与MeJA提高水稻相关防御酶的活性有关.     

云南文山州首次在感病西葫芦上检测到番木瓜环斑病毒

为明确引起云南文山州广南县西葫芦发生蕨叶、斑驳、泡状斑等症状的病原,从采集的西葫芦病样中提取总RNA,利用RT-PCR扩增后经电泳检测及测序获得大小为887bp的片段;经过BLAST序列同源性比对分析,该病原与番木瓜环斑病毒Papaya ringspot virus(PRSV)(KY996464)基因序列同源性达87%。由此可知,侵染文山州西葫芦的病毒病原是番木瓜环斑病毒。     

茉莉酸甲酯诱导大田水稻抗白叶枯病的效应研究

本研究以感白叶枯病水稻品种‘温229’为试验材料,通过茉莉酸甲酯(MeJA)浸种和喷雾处理幼苗期和孕穗期水稻,探索MeJA诱导大田水稻抗白叶枯病的效应。水稻幼苗期,0.004mmol/L MeJA浸种、0.1mmol/L MeJA喷雾、0.004mmol/L MeJA浸种后再用0.1mmol/L MeJA喷雾处理,均表现诱导幼苗抗白叶枯病的作用,其诱导效果最高达27.65%;其中,MeJA浸种再喷雾双重处理的效果与单一喷雾处理差异不显著,但与单一浸种处理达到显著性差异水平;水稻孕穗期,0.05~2.00mmol/L的MeJA喷雾后均可降低白叶枯病的病情指数,其中2011年MeJA诱导效果最佳的浓度为0.50mmol/L,而2012年和2013年则为1.00mmol/L,诱导效果最高为17.17%。以上结果表明,MeJA处理可有效缓解大田水稻幼苗和孕穗期白叶枯病的发生,其诱导效果可能与MeJA处理浓度、方法及水稻生育期有关。     

侵染冬瓜的病毒病原种类鉴定

本研究在我国主要冬瓜产区采集具有典型病毒病症状的病叶材料105份,根据葫芦科作物上常见的5种病毒病原的CP基因设计特异性引物,对105份待检冬瓜材料进行RT-PCR检测。检测结果表明:5对特异引物可分别在105份待检材料的95份中检测到小西葫芦黄花叶病毒(Zucchini yellow mosaic virus,ZYMV)、西瓜花叶病毒(Watermelon mosaic virus,WMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番木瓜环斑病毒(Papaya ringspot virus,PRSV)4种病毒,未检测到南瓜花叶病毒(Squash mosaic virus,SqMV);并且发现不同的冬瓜主产区致病的病毒种类有较大差异;同时还发现,在这些待检样品中4种病毒复合侵染现象较普遍,其中以PRSV与WMV组合最常见,占复合侵染现象的31.25%;未发现有4种病毒复合侵染。     

Transmission efficiency of Papaya ringspot virus by three aphid species

The transmission efficiency of Papaya ringspot virus (PRSV) by three aphid vectors (i.e., Aphis gossypii, A. craccivora, and Myzus persicae) was studied. Efficiency was measured by single-aphid inoculation, group inoculation (using five aphids), duration of virus retention, and the number of plants following a single acquisition access period (AAP) to which the aphids could successfully transmit the virus. Single-aphid inoculation studies indicated that M. persicae (56%) and A. gossypii (53%) were significantly more efficient in transmitting PRSV than A. craccivora (38%). Further, in the former two species, the time required for initiation of the first probe on the inoculation test plant was significantly shorter compared to A. craccivora. PRSV transmission efficiency was 100% in all three species when a group of five aphids were used per plant. There was a perceptible decline in transmission efficiency as the sequestration period increased, although M. persicae successfully transmitted PRSV after 30 min of sequestration. A simple leaf-disk assay technique was employed for evaluating the transmission efficiency of three species of aphids. The results of leaf-disk assays also indicated that A. gossypii (48%) and M. persicae (56%) were more efficient PRSV vectors than A. craccivora. Using leaf-disk assays, the ability of individual aphids to inoculate PRSV serially to a number of plants was studied. Following a single AAP on an infected leaf, M. persicae was more efficient than the other two species with 52.5% transmission after the first inoculation access period (IAP). However, its inoculation efficiency significantly decreased with the second and subsequent IAPs. A. gossypii was able to transmit PRSV sequentially up to four successive leaf disks, but with significantly declining efficiency. Since A. gossypii is reported to be the numerically dominant vector in south India in addition to being a more efficient vector capable of inoculating PRSV to multiple plants, it should be the target vector for control strategies.     

Molecular characterization of two papaya ringspot virus isolates that cause devastating symptoms in Norte de Santander,Colombia

Papaya ringspot virus is an RNA virus that belongs to the genus Potyvirus, family Potyviridae and affects both papaya and cucurbits, causing great economic losses. PRSV isolates are divided into biotypes P and W; both biotypes naturally infect plants in the family Cucurbitaceae, whereas the P type also naturally infects papaya (Carica papaya L). In the present study, we report the full-length genome sequence of two PRSV-P isolates sampled from the Campo Hermoso (PRSV-CH) and Villa del Rosario (PRSV-VR) localities in Norte de Santander, Colombia. The genomes of these PRSV isolates are 10,326 nt in length and have a predicted ORF of 3344 aa. The identity among Colombian PRSV isolates is 96.9% and 97.3% at the nucleotide and deduced amino acid levels, respectively. PRSV isolates from China had the lowest identity at 78.3% and 89.2% (nucleotide-amino acid), whereas the highest identities were detected in PRSV isolates from Mexico, Venezuela and Hawaii. At the polyprotein level, the amino acid composition surrounding the active polyprotein cleavage sites differ in the Colombian PRSV sequences. The predicted cleavage site in P1/HC-Pro is LEQY/N – LEQY/S instead of MEQY/N. Both of the Colombian PRSV isolates have a putative recombination event in the P1 coding region, which is common in all PRSV isolates from the American continent. The new full-length PRSV sequences from Colombia provide a better understanding of the dynamics of papaya ringspot virus infections in papaya in Colombia and worldwide.     

Suppression of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> infection in <Emphasis Type="Italic">Carica papaya</Emphasis> with CAP-34, a systemic antiviral resistance inducing protein from <Emphasis Type="Italic">Clerodendrum aculeatum</Emphasis>

CAP-34, a protein from Clerodendrum aculeatum inducing systemic antiviral resistance was evaluated for control of Papaya ringspot virus (PRSV) infection in Carica papaya. In control plants (treated with CAP-34 extraction buffer) systemic mosaic became visible around 20 days that intensified up to 30 days in 56% plants. During this period, CAP-34-treated papaya did not show any symptoms. Between 30 and 60 days, 95% control plants exhibited symptoms ranging from mosaic to filiformy. In the treated set during the same period, symptoms appeared in only 10% plants, but were restricted to mild mosaic. Presence of PRSV was determined in induced-resistant papaya at the respective observation times by bioassay, plate ELISA, immunoblot and RT-PCR. Back-inoculation with sap from inoculated resistant plants onto Chenopodium quinoa did not show presence of virus. The difference between control and treated sets was also evident in plate-ELISA and immunoblot using antiserum raised against PRSV. PRSV RNA was not detectable in treated plants that did not show symptoms by RT-PCR. Control plants at the same time showed a high intensity band similar to the positive control. We therefore suggest that the absence/delayed appearance of symptoms in treated plants could be due to suppressed virus replication.     

Transgene-mediated resistance to <Emphasis Type="Italic">Papaya ringspot virus</Emphasis>: challenges and solutions

Global papaya production is severely affected by papaya ringspot disease caused by Papaya ringspot virus (PRSV). Management of this potyvirus is challenging, due to 1) its non-persistent transmission by numerous aphid species and 2) the diversity of PRSV strains that exists within a country or between different geographical regions. Papaya cultivars with transgenic resistance have reduced the impact of the disease. There are no effective alternatives to transgenic resistance available in areas where disease pressure is high. In Hawaii, transgenic papayas such as “SunUp” and “Rainbow” have remained resistant to PRSV more than two decades saving the commercial papaya industry. Following the success in Hawaii, researchers from other countries have focused on developing PRSV-resistant transgenic papaya. These transgenic cultivars often demonstrated an initial transitory resistance that was ultimately overcome by the virus. For other cases, resistance was inconsistent. That is, some transgenic lines were resistant while others were not. Transgenic cultivars are now losing PRSV-resistance for various reasons in China and Taiwan. In this review, we present an update on work with transgenic papaya with resistance to PRSV. The focus is on factors affecting transgenic resistance in papaya and our attempt to explain why the Hawaiian scenario of complete and durable resistance has not been replicated in other regions. The utilization of more recent technologies to the development of virus resistance in papaya is also discussed.     

Engineered Mild Strains of Papaya ringspot virus for Broader Cross Protection in Cucurbits

ABSTRACT Papaya ringspot virus (PRSV) HA5-1, a mild mutant of type P Hawaii severe strain (PRSV P-HA), has been widely used for the control of PRSV type P strains in papaya, but did not provide practical protection against PRSV type W strains in cucurbits. In order to widen the protection effectiveness against W strains, chimeric mild strains were constructed from HA5-1 to carry the heterologous 3' genomic region of a type W strain W-CI. Virus accumulation of recombinants and their crossprotection effectiveness against W-CI and P-HA were investigated. In horn melon and squash plants, the recombinant carrying both the heterologous coat protein (CP) coding region and the 3' untranslated region (3'UTR), but not the heterologous CP coding region alone, significantly enhanced the protection against W-CI. The heterologous 3'UTR alone is critical for the enhancement of the protection against W-CI in horn melon, but not in zucchini squash. In papaya, the heterologous CP coding region or 3'UTR alone, but not both together, significantly reduced the effectiveness of cross protection against P-HA. Our recombinants provide broader protection against both type W and P strains in cucurbits; however, the protective effectiveness is also affected by virus accumulation, the organization of the 3' genomic region, and host factors.     

The Complete Nucleotide Sequence and Biotype Variability of Papaya leaf distortion mosaic virus

ABSTRACT The complete nucleotide sequence of the genome of Papaya leaf distortion mosaic virus (PLDMV) was determined. The viral RNA genome of strain LDM (leaf distortion mosaic) comprised 10,153 nucleotides, excluding the poly(A) tail, and contained one long open reading frame encoding a polyprotein of 3,269 amino acids (molecular weight 373,347). The polyprotein contained nine putative proteolytic cleavage sites and some motifs conserved in other potyviral polyproteins with 44 to 50% identities, indicating that PLDMV is a distinct species in the genus Potyvirus. Like the W biotype of Papaya ringspot virus (PRSV), the non-papaya-infecting biotype of PLDMV (PLDMV-C) was found in plants of the family Cucurbitaceae. The coat protein (CP) sequence of PLDMV-C in naturally infected-Trichosanthes bracteata was compared with those of three strains of the P biotype (PLDMV-P), LDM and two additional strains M (mosaic) and YM (yellow mosaic), which are biologically different from each other. The CP sequences of three strains of PLDMV-P share high identities of 95 to 97%, while they share lower identities of 88 to 89% with that of PLDMV-C. Significant changes in hydrophobicity and a deletion of two amino acids at the N-terminal region of the CP of PLDMV-C were observed. The finding of two biotypes of PLDMV implies the possibility that the papaya-infecting biotype evolved from the cucurbitaceae-infecting potyvirus, as has been previously suggested for PRSV. In addition, a similar evolutionary event acquiring infectivity to papaya may arise frequently in viruses in the family Cucurbitaceae.     

番木瓜环斑病毒西瓜株系弱毒突变体的筛选与应用

为防治番木瓜环斑病毒(Papaya ringspot virus,PRSV)在我国葫芦科作物上引起的病毒病害,以PRSV西瓜株系山东分离物(PRSV-SD)侵染性cDNA克隆为基础,采用定点突变方法将辅助成分-蛋白酶保守氨基酸137位和346位的天冬酰胺(N)和417位的缬氨酸(V)突变为丙氨酸(A),应用农杆菌浸润法接种西葫芦叶片并分析突变对PRSV-SD致病力的影响,筛选弱毒突变体,进而评价其交叉保护效果。结果表明,与野生型PRSV-SD相比,获得的3个突变体N137A、N346A和V417A,接种后在西葫芦植株上的症状明显减轻,衣壳蛋白在叶片中的积累水平分别为野生型PRSV-SD的24.0%、13.0%和4.0%,均为弱毒突变体。当保护间隔期为10 d时,弱毒突变体N137A具有完全的交叉保护效果,N346A可延迟发病15 d,而V417A无交叉保护效果。当间隔保护期为15 d时,弱毒突变体N137A和N346A的保护效率分别为100.0%和26.7%,而V417A无交叉保护效果。