CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR–Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: ‘San Marzano’ (SM) and ‘Oxheart’ (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a −7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (−7 bp and −2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.     

烟草低头黑病菌毒素钝化物的初步研究

研究了10种化合物4个浓度对烟草低头黑病菌(Colletotrichum capsici f.nicotianae)毒素的钝化作用。结果表明，高锰酸钾、氢氧化钠和碳酸氢钠对毒素具有很强的钝化作用。高锰酸钾在离体叶片上的相对防效为100％；各浓度的氢氧化钠和碳酸氢钠均对烟草低头黑病菌菌丝生长和分生孢子萌发具有一定的抑制作用。高锰酸钾并不抑制菌丝生长。     

四川凉山地区烟草黑胫病菌的ITS序列分析

本研究利用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增四川省凉山州不同地区的20株烟草黑胫病菌的ITS序列,并对PCR产物进行了克隆测序。结果表明,20个供试菌株的ITS1-5.8S-ITS2总长均为803 bp;各菌株之间的ITS序列同源性达到99.3%以上;与GenBank报道的寄生疫霉台湾烟草分离株Phytophthora parasitica(GU111675.1)的同源性在99.6%~100%之间;但各菌株ITS序列的个别位点存在突变,这些突变主要集中在ITS2区上,其中第468处的突变具有一定地域性,主要为德昌和西昌的菌株。利用MEGA6软件对20个供试菌株及GenBank中登陆的15个疫霉属菌株序列进行聚类分析,供试菌株与GenBank上登录的GU111675.1、KF010303.1、AJ854295.1、KC768775.1、L41383聚在同一聚类组上,均为Phytophthora parasitica(或Phytophthora nicotianae),其它疫霉种聚在不同的聚类组上,遗传关系较远,这表明来自四川凉山不同地区的20株烟草黑胫病分离菌株均为寄生疫霉(Phytophthora parasitica)。     

辣椒疫霉对杀菌剂抗药性研究进展

综述了近年来国内外辣椒疫霉菌对苯基酰胺类、甲氧基丙烯酸酯类、丙烯酰胺类内吸性杀菌剂的抗药性发生现状、抗药机理、抗药遗传等方面的研究进展.对辣椒疫霉菌抗药性研究和制定抗药性治理对策以及化学防治具有一定的参考价值,同时指出了今后的研究方向和发展前景.     

Optimal strategies to protect a sub-population at risk due to an established epidemic

Epidemics can particularly threaten certain sub-populations. For example, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the elderly are often preferentially protected. For diseases of plants and animals, certain sub-populations can drive mitigation because they are intrinsically more valuable for ecological, economic, socio-cultural or political reasons. Here, we use optimal control theory to identify strategies to optimally protect a ‘high-value’ sub-population when there is a limited budget and epidemiological uncertainty. We use protection of the Redwood National Park in California in the face of the large ongoing state-wide epidemic of sudden oak death (caused by Phytophthora ramorum) as a case study. We concentrate on whether control should be focused entirely within the National Park itself, or whether treatment of the growing epidemic in the surrounding ‘buffer region’ can instead be more profitable. We find that, depending on rates of infection and the size of the ongoing epidemic, focusing control on the high-value region is often optimal. However, priority should sometimes switch from the buffer region to the high-value region only as the local outbreak grows. We characterize how the timing of any switch depends on epidemiological and logistic parameters, and test robustness to systematic misspecification of these factors due to imperfect prior knowledge.     

大豆资源抗疫霉根腐病基因分析

采取下胚轴创伤接种法鉴定156份大豆资源对13个不同毒力基因型大豆疫霉菌株的抗性。结果表明,125份资源分别抗1～13个菌株,占鉴定资源总数的80.13%。125份抗性大豆资源与13个大豆疫霉菌株共产生90种反应型。通过与13个鉴别寄主的反应型比较发现,有9份大豆资源产生的5种反应型与含有已知抗病基因的大豆资源的反应型相同;12份大豆资源产生的5种反应型与已知2个抗病基因组合的反应型一致,另外,还有至少抗1个菌株的104份大豆资源产生的80种反应型,既不同于已知单个抗病基因的反应型,也不同于2个已知抗病基因组合的反应型,推测可能含有新的抗病基因或基因组合。     

黑龙江省大豆品种对大豆疫霉根腐病抗性评价及抗性基因推导

采用下胚轴伤口接种法,利用课题组在黑龙江省分离鉴定的8个大豆疫霉根腐病生理小种（race1、race3、race4、race5、race9、race13、race44、race54）对黑龙江省曾经大面积推广种植的44个大豆品种进行接种鉴定。结果表明,对8个生理小种分别有45.5%、29.5%、40.9%、43.2%、36.8%、45.5%、31.8%、72.7%表现抗性或中间类型,共产生37种反应型;通过基因推导,合丰-44,绥农-12可能含有Rps1c基因或Rps1a＋Rps1c、Rps1c＋Rps6、Rps1c＋Rps7的基因组合;黑河-32可能含有Rps1a＋Rps1d的基因组合,其它品种可能含有新的抗病基因或基因组合,表明黑龙江省存在丰富的抗大豆疫霉根腐病的种质资源。     

蛇床子素结构修饰物JS-B对辣椒疫霉生物活性

对蛇床子素结构修饰物JS-B的生物活性进行了研究.室内生测结果表明:JS-B对辣椒疫霉菌丝干重和游动孢子萌发的抑制中浓度(EC50)分别为43.74、86.03 mg/L.盆栽试验表明:JS-B处理对辣椒疫病有一定预防和治疗效果.根系对JS-B具有一定的吸收性,1000 mg/L JS-B灌根处理,防效达67.16%.用400 mg/L的JS-B喷雾处理辣椒幼苗,分别在施药后1、3、8 d接种辣椒疫霉的游动孢子,其对辣椒疫病的防效分别为71.95%、61.90%和66.72%,与阳性对照药剂烯酰吗啉相当,说明JS-B在防治辣椒疫病中具有一定的持效期.     

土壤中烟草疫霉的分离与量化测定

从温室盆栽接种试验土和烟田土中分别取样，在室温下晾干，研碎并通过１ｍｍ直径的网筛，用选择性培养基土壤稀释平板法和叶片诱饵法分离烟草疫霉菌株；用土壤稀释平板法测定了云南主要烟区土样的烟草疫霉密度。结果看出，连作烟田的病菌密度高于轮作田，低凹烟田高于山地烟田。叶片诱饵法试验表明，以烟茎切片的诱集效果最好，其次是烟叶和马铃薯叶，番茄叶和辣椒叶的诱发效果相似，花生叶诱集不到烟草疫霉。