Assessing the severity of cotton Verticillium wilt disease from in situ canopy images and spectra using convolutional neural networks

Verticillium wilt(VW)is a common soilborne disease of cotton.It occurs mainly in the seedling and bollopening stages and severely impairs the yield and quality of the fiber.Rapid and accurate identification and evaluation of VW severity(VWS)forms the basis of field cotton VW control,which has great significance to cotton production.Cotton VWS values are conventionally measured using in-field observations and laboratory test diagnoses,which require abundant time and professional expertise.Remote and proximal sensing using imagery and spectrometry have great potential for this purpose.In this study,we performed in situ investigations at three experimental sites in 2019 and 2021 and collected VWS values,in situ images,and spectra of 361 cotton canopies.To estimate cotton VWS values at the canopy scale,we developed two deep learning approaches that use in situ images and spectra,respectively.For the imagery-based method,given the high complexity of the in situ environment,we first transformed the task of healthy and diseased leaf recognition to the task of cotton field scene classification and then built a cotton field scenes(CFS)dataset with over 1000 images for each scene-unit type.We performed pretrained convolutional neural networks(CNNs)training and validation using the CFS dataset and then used the networks after training to classify scene units for each canopy.The results showed that the Dark Net-19 model achieved satisfactory performance in CFS classification and VWS values estimation(R^(2)=0.91,root-mean-square error(RMSE)=6.35%).For the spectroscopy-based method,we first designed a one-dimensional regression network(1D CNN)with four convolutional layers.After dimensionality reduction by sensitive-band selection and principal component analysis,we fitted the 1D CNN with varying numbers of principal components(PCs).The 1D CNN model with the top 20 PCs performed best(R^(2)=0.93,RMSE=5.77%).These deep learning-driven approaches offer the potential of assessing crop disease severity from spatial and spectral perspectives.

Efficacy evaluation and mechanism of Bacillus subtilis EBS03 against cotton Verticillium wilt

Background:In our previous study,a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt.However,its mechanism for controlling Verticillium wilt remains unclear.The objective of this study was to further clarify its con-trolling effect and mechanism against cotton Verticillium wilt.Results:The results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03%and 59.00%,respectively;the inhibitory effects of sporulation and microsclerotia germination were 47.16%and 70.06%,respec-tively.In the greenhouse test,the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11%on cotton Verticillium wilt,and significantly promoted the growth of cotton seedlings.In the field experi-ment,the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54%at 60 days after cotton sowing,and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking,root irrigation,and spraying treatments significantly increased by 19.48%and 7.42%,30.90%and 2.62%,15.99%and 9.20%,respec-tively.Furthermore,EBS03 improved the resistance of cotton leaves against the infection of V.dahliae,and induced the outbreak of reactive oxygen species and accumulation of callose.In addition,the results of real time fluorescent quantitative polymerase chain reaction(RT-qPCR)detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL,POD,PPO,and PR10 in cotton leaves,enhanced cotton plant resistance to V.dahliae,and inhibited colonization level of this fungal pathogen in cotton.Conclusion:Bacillus subtilis EBS03 has a good biological defense capability,which can inhibit the growth and coloni-zation level of V.dahliae,and activate the resistance of cotton to Verticillium wilt,thus increase cotton yield.

Study on Cotton Verticillium Wilt

The pathogen, characteristics, pathogenic mechanism, infection cycle, occurrence regularity and damage symptom of cotton Verticillium wilt are expounded in the paper. The paper puts forward the strategy of protecting disease-free area first, planting resistant varieties and rational rotation in severe area and strengthening the field management, as well as combining with biological control and chemical control methods to effectively control infection and spread of pathogen, so as to provide certain theoretical basis for reducing the damage of cotton Verticillium wilt and improving yield and quality of cotton.

Physiological mechanisms involved in resistance to cotton verticillium wilt induced by AM fungi

It was proved that arbuscular mycorrhizal (AM) fungi played an important role in increasing plant resistance to soilborne pathogens, especially when plants were pre-inoculated with AM fungi. Mechanisms involved in this phenomenon are not yet well understood. On the basis of the former experiment results in our lab, effects of AM fungi on cotton Verticillium wilt and the mechanisms of increasing disease resisitance by the tested fungi were studied in pot culture under greenhouse conditions. Two cotton cutivars Litai 8 and 86-1 which are susceptible to Verticillium dahliae were pre-inoculated with Glomus fasiculatum, and Gigaspora margarita, then inoculated with the strain of Verticillium dahliae, namely “An-Yang” (belong to intermediate virulent type) 30 days after the former inoculation. Results showed that AM fungi could improve the growth and development of cotton plants, increase plants dry mass, decrease incidence and disease index of Verticillium wilt of cotton plants, inhibit the infection and development of V. dahliae to different extent in the rhizosphere of cotton pre-inoculated with AM fungi, while the colonization and spore numbers of AM fungi were not reduced significantly by this pathogen. The defence enzymes, such as phenylalanine ammonia-lyase (PAL), chitinase, β-1,3-glucanase, peroxidase, polyphenoloxidase (PPO) were induced, and their activities and peak increased by AM fungi in roots and leaves, and the increasing speed and peak of the enzyme activity were higher in treatment with AM fungus preinoculation than the inoculation with only V. dahliae, which suggested that defense response was activated by AM fungi, and then made the cotton to react strongly and rapidly to the infection of V. dahliae. In addition, AM fungi decreased the content of malondiadehyde (MDA) in cotton roots and leaves, protected membrane system and alleviated the damage caused by the pathogen. The AM fungus, Glomus fasiculatum showed the superior effects of biological control. It was concluded that AM fungi could provide the biological control to cotton diseases and there were application prospects of this biological agents.

Development and identification of Verticillium wilt-resistant upland cotton accessions by pyramiding QTL related to resistance

Cotton Verticillium wilt is a serious soil-borne disease that leads to significant losses in fiber yield and quality worldwide. Currently, the most effective way to increase Verticillium wilt resistance is to develop new resistant cotton varieties. Lines 5026 and 60182 are two Verticillium wilt-resistant upland cotton accessions. We previously identified a total of 25 quantitative trait loci（QTLs） related to Verticillium wilt resistance from 5026 and 60182 by assembling segregating populations from hybridization with susceptible parents. In the current study, using 13 microsatellite markers flanking QTLs related to Verticillium wilt resistance, we developed 155 cotton inbred lines by pyramiding different QTLs related to Verticillium wilt resistance from a filial generation produced by crossing 5026 and 60182. By examining each allele＇s effect and performing multiple comparison analysis, we detected four elite QTLs/alleles（q-5/NAU905-2, q-6/NAU2754-2, q-8/NAU3053-1 and q-13/NAU6598-1） significant for Verticillium wilt resistance, pyramiding these elite alleles increased the disease resistance of inbred lines. Furthermore, we selected 34 elite inbred lines, including five lines simultaneously performing elite fiber quality, high yield and resistance to V. dahliae, 14 lines with elite fiber quality and disease resistance, three lines with high yield and disease resistance, and 12 lines with resistance to V. dahliae. No correlation between Verticillium wilt resistance and fiber quality traits/yield and its components was detected in the 155 developed inbred lines. Our results provide candidate markers for disease resistance for use in marker-assisted breeding（MAS）, as well as elite germplasms for improving important agronomic traits via modern cotton breeding.

Advances and prospects of genetic mapping of Verticillium wilt resistance in cotton

Verticillium wilt is one of the most important diseases affecting cotton production in China.The fungus,Verticillium dahliae,has a wide host range and a high degree of genetic variability.No resistance resources have been found in the available planting resources,thus presenting difficulties and challenges for our study.The long-term production practice shows that selection of disease-resistant varieties is the most economical and effective measure to control Verticillium wilt of cotton to reduce the yield loss and quality decline of cotton.In this paper,we summarized the genetic mapping population,the analysis method of genetic localization,the discovery,mining and cloning of disease-resistant quantitative trait loci/markers,and the analysis of their genetic functions,so as to provide information for the molecular breeding approach of disease-resistant cotton.

Spectrum Characteristics of Cotton Canopy Infected with Verticillium Wilt and Applications

Hyper spectrum remote sensing with fine spectrum information is an efficient method to estimate the verticillium wilt of cotton. The research was conducted in Xinjiang, the largest cotton plant region of China, by using the data which were collected both by canopy spectrum infected with verticillium wilt and severity level （SL） in the year 2005-2006. The quantitative correlation was analyzed between SL and canopy of reflectance spectrum or derivative spectrum reflectance. The results indicated that spectrum characteristics of cotton canopy infected with verticillium wilt changed regularly with the increase of SL in different periods and varieties, Spectrum reflectance increased in the visible light region （620-700 nm） with the increase of the SL, which inverted in near-infrared region and was extremely significant in the region of （780-1 300 nm）. When SL attained b2 （DI = 25）, cotton canopy infected with verticillium wilt was used as a watershed and diagnosed index in the beginning stages of the disease. The results also indicated that there were marked different characteristics of the first derivative spectrum in these SL, it changed significantly in the red edge ranges （680-760 nm） with different SL, i.e., red edge swing decreased, and red edge position equally moved to the blue. In this study 1 001-1 110 nm and 1 205- 1 320 nm were selected out as sensitive bands for SL of canopy. Inversion models established for estimating cotton canopy infected with verticillium wilt reached the most significant level. Finally, the different spectrum characteristics of cotton canopy infected with verticillium wilt were marked, some inversion models were established, which could estimate SL of canopy infected with verticillium wilt. The best recognized model was the first derivative spectra at （FD 731 nm- FD 1317 nm）, and it might be used to forecast the position of cotton canopy infected with verticillium wilt quantitatively.

Advances in Cotton Breeding for Resistance to Fusarium and Verticillium Wilt in the Last Fifty Years in China

This review summarizes the main advances in cotton breeding for resistance to fusarium and verticillium wilt in the last fifty years in China. The topics discussed include main achievements, experiences, problems and countermeasures for solving.

Isolation and characterization of the GbVIP1 gene and response to Verticillium wilt in cotton and tobacco

Background: Verticillium wilt is a serious soil-borne vascular disease that causes major losses to upland cotton(Gossypium hirutum L.) worldwidely every year. The protein VIP1(VirE2 interaction protein 1), a bZIP transcription factor, is involved in plant response to many stress conditions, especially pathogenic bacteria. However, its roles in cotton response to Verticillium wilt are poorly understood.Results: The GbVIP1 gene was cloned from resistant sea-island cotton(G. barbadense) cv. Hai 7124. Expression of GbVIP1 was up-regulated by inoculation with Verticillium dahliae and exogenous treatment with ethylene. Results of virus-induced gene silencing suggested that silencing of GbVIP1 weakened cotton resistance to Verticillium wilt. The heterologous expression of GbVIP1 in tobacco showed enhanced resistance to Verticillium wilt. The PR1, PR1-like and HSP70 genes were up-regulated in GbVIP1 transgenic tobacco after Verticillium wilt infection.Conclusion: Our results suggested that GbVIP1 increased plant resistance to Verticillium wilt through up-regulating expressions of PR1, PR1-like, and HSP70. These results provide new approaches to improving resistance to Verticillium wilt in upland cotton and also have great potential for disease-resistance breeding of cotton.

Studies on Cotton Breeding Resistant to Fusarium and Verticillium wilt Diseases

Both Fusarium and Verticillium wilts are important soil-borne diseases,which can not be effectively controlled by chemical fungicides.The two diseases,especially Verticillium wilt,have

Test on Wilt-Resistance of Simple Hybrides F7 and Beckross Hybrid Lines F6 B1 of <i>G. hirsutum</i>L. of Cotton to New Virulent Isolates of Fungus <i>Fusarium verticillioides</i>and <i>Verticillium dahliae</i>

The article presents the results of studies on the resistance of hybrid cotton lines to a new virulent isolate (strain) of the fungus Fusarium verticillioides upon inoculation of the host plant. Based on the studies, it was found that the complex genotypic resistance of the studied lines, when the host plants are inoculated with isolates of -100 V. dahliae Kleb fungus and 103 Fusarium verticillioides fungi, depends on the degree of resistance of the parental forms and their combination ability.

35份棉花品系对黄萎病抗性鉴定及综合分析

【目的】筛选出品质优、产量高和抗病性强的棉花品系。【方法】以35份棉花品系为研究对象,对该群体18个主要农艺性状进行主成分和聚类分析。【结果】35份棉花品系的各农艺性状变异系数范围为1.67%(整齐度,UI)~43.10%(相对病情指数,IR),其中相对病情指数(IR)、铃数(TBN)、有效铃数(EBN)和单株皮棉产量(LYPP)变异系数较大,1号(4-13)、2号(4-22)、10号(4-2-7-3)和16号(10Q-67-3)相对病情指数小于20,4个品系对黄萎病的抗性较强;单株皮棉产量与各性状之间相关性不一,与品质性状之间也存在制约;5个主成分特征根值分别为5.334、3.423、3.105、1.298和1.105,贡献率分别为29.636%、19.019%、17.249%、7.214%和6.141%,累计贡献率达到79.259%;将35份棉花品系分为四类,第I类和第Ⅱ类品系综合表现为品质优异,抗病性强类群,但产量低;第Ⅲ类品系综合表现为高产类群,但品质差,抗病性弱;第Ⅳ类品系综合表现为耐病性类群,但产量略低,品质略差。【结论】将各类群与主成分综合得分F值排名前10取交集,优异品系为21号(37-129-3)和30号(44-17-8)。

棉花抗黄萎病生理生化机制研究进展

棉花是我国的重要经济作物,是关系我国国计民生的支柱性产业,是影响世界大宗农产品及下游纺织品贸易格局的战略性产业。由大丽轮枝菌Verticillium dahliae引起的棉花黄萎病是棉花生产上的头号病害,严重影响棉花产量与质量。黄萎病菌侵染过程中会激发棉花植株产生一系列防御反应,如组织结构抗性变化、植物激素信号通路响应及抗病相关基因表达等。目前,已从棉花中挖掘出大量响应黄萎病菌的基因,阐明了这些抗性相关基因通过调控生理生化反应而发挥抗病功能的机制。本文从活性氧、防御酶、次生代谢产物和植物激素4个方面综述了棉花抗性相关生理生化作用机制,总结了棉花抗性相关生理生化反应的调控网络,为深入了解棉花抗黄萎病机制、选育抗病品种提供理论依据。

大丽轮枝菌(Verticillium dahliae)微菌核缺陷突变体的筛选及其侧端序列分析

为了获得大丽轮枝菌(Verticillium dahliae)微菌核缺陷突变体,利用农杆菌介导转化大丽轮枝菌,共获得270个T-DNA随机插入突变体,从中筛选出了4个气生菌丝发达、不产微菌核的突变体,利用高效TAIL-PCR技术,获得了其侧端序列,结合生物信息学方法,获得4个突变体T-DNA在大丽轮枝菌基因组插入位点的基因信息。

大丽轮枝菌(Verticillium dahliae)酯酶同工酶的测定

对棉花黄萎病菌不同致病力及致病类型菌系，不同寄主上的大力轮枝菌（Verticilliumdahliae）共14个菌系的酯酶，进行了聚丙烯酰胺凝胶平板电泳。结果表明棉花黄萎病不同致病类型菌系有其独特的酯酶同工酶谱。E6酶带的有无与致病力类型有关，E3酶带活性与病菌致病力有关，E3酶带活性与棉花发病病指关联度为0．85。不同寄主大丽轮枝菌酯酶同工酶总酶带数在7～12条之间，主酶带数在2～4条之间，按E3酶带活性强、中、弱划分为3个类型。E3酶带活性与病菌致病力有关，其活性大小与棉花、番茄、茄子病指的关联度均为0．774以上。说明酯酶同工酶技术可和于鉴定大丽轮枝菌同一种内不同致病类型致病力菌系。

棉花品种资源黄萎病田间抗性鉴定和评价

通过田间抗病性鉴定和分析,掌握种质抗病性分布状况,筛选抗黄萎病核心资源,经遗传改良,定向选育与新疆棉花产业需求相吻合的抗病品种,并探讨病害治理策略。以收集的396份陆地棉品种资源为鉴定对象,2018-2019年,通过自然病田小区试验,在6月底(蕾期)和8月底(铃期),采用5级分级法,对品种资源进行黄萎病抗病性鉴定和评价。供试陆地棉种质具备丰富的抗病遗传多样性,种质间黄萎病变异系数高,可选择抗病种质加以利用。年度间各材料发病程度差异大,2018年发病明显重于2019年,但材料间发病趋势基本相同;蕾期发病较轻,铃期发生重。两年自然病田鉴定平均73.3%的材料蕾期未发病;铃期相对病指超过20的材料占比46.0%,相对病指为0.1~20.0的占比50.4%,未发病的占3.6%。铃期发病最重材料的发病率达100%,病指高达75.0;2018年铃期参加鉴定材料平均发病率和病指分别达38.8%、19.2,比2019年分别高19.0个百分点和8.7。发病率与病指间存在极显著正相关,相关系数>0.93;蕾期发病率和病指比铃期变异系数大,蕾期与铃期存在极显著正相关,平均相关系数分别为0.28、0.32。鉴定出36份抗黄萎病优异资源(自然病田铃期黄萎病病指≤10、纤维长度≥29 mm、断裂比强度≥29.0 cN/tex、马克隆值3.5~4.9、小区皮棉产量≥2250 kg/hm^(2)),这些资源地域来源较广,抗黄萎能力较强,与产量、品质匹配较好,可在抗病育种遗传改良中利用。

基于F2和RIL群体鉴定棉花抗黄萎病主效QTL

【目的】通过对棉花抗黄萎病性状进行数量性状位点(quantitative trait locus,QTL)定位,鉴定可以应用于育种实践的能稳定检测到的主效QTL,为棉花抗黄萎病遗传改良奠定分子基础。【方法】以抗黄萎病品种中植棉2号和感黄萎病品种冀棉11号为亲本杂交的F2群体和重组自交系(recombinant inbred lines,RIL)群体作为作图群体,在对2个群体进行多环境黄萎病抗性鉴定和简单重复序列(simple sequence repeat,SSR)分子标记检测的基础上进行遗传连锁图谱构建,利用完备区间作图法进行QTL定位,并对获得的主效QTL置信区间进行候选基因挖掘。【结果】在F2:3家系和RIL群体中共检测到7个抗黄萎病QTL,能够在多个环境条件下重复检测到的QTL有4个,包括q VW-D05-1、qVW-D05-2、q VW-D05-4和qVW-D05-5。共线性分析表明上述4个QTL集中分布于D05染色体上2293776~3205058 bp和62407897~62582344 bp 2个区域。4个抗性QTL的聚合可以显著降低棉花黄萎病病情指数。qVW-D05-1在多个环境中的表型变异解释率均在10%以上,为抗黄萎病主效QTL。对qVW-D05-1置信区间内的基因进行了功能注释、黄萎病菌诱导后的表达模式分析和单核苷酸多态性(single nucleotide polymorphism,SNP)变异分析,初步推测Ghir_D05G038990、Ghir_D05G039060、Ghir_D05G039100、Ghir_D05G039110和Ghir_D05G039130为可能控制棉花黄萎病抗性的候选基因。【结论】在不同环境中共检测到7个抗黄萎病QTL,其中4个QTL被重复检测到,在主效QTL q VW-D05-1区间内筛选获得5个棉花抗黄萎病候选基因。这些稳定的抗黄萎病相关的QTL及其候选基因可应用于棉花抗黄萎病性状的分子标记辅助选择育种。

基于西兰花尾菜为载体的解淀粉芽胞杆菌PHODG36颗粒剂研制及其对棉花黄萎病的防治效果

本研究以防病促生的解淀粉芽胞杆菌PHODG36为有效成分、西兰花尾菜干粉和草炭为载体研制出解淀粉芽胞杆菌颗粒剂,在温室和田间小区条件下,研究该颗粒剂对棉花出苗和植株生物量的影响,并评价其对棉花黄萎病的防治效果。结果表明,制备的颗粒剂外观为黑色、短柱状、平均长度为8 mm,芽胞含量为1.5×10^(9)CFU/g。与空白对照相比,培养基质中添加0.1%-0.5%颗粒剂的校正出苗率差异不显著,而随着颗粒剂用量的增加(1%和2%),棉花校正出苗率呈显著下降趋势。颗粒剂用量0.3%处理对棉花株高、植株鲜重和干重的促生率分别为14.53%、16.38%和17.05%。综合考虑上述因素,确定颗粒剂最佳用量为0.3%;温室防病试验表明,土壤中添加0.3%颗粒剂对棉花黄萎病的防治效果为61.60%。采用田间撒施法研究颗粒剂在500、1000和1500 kg/667 m^(2)剂量下对黄萎病的防治效果,结果表明该菌剂500 kg/667 m^(2)对黄萎病的防治效果最高,达到67.28%。综合分析表明,解淀粉芽胞杆菌颗粒剂能够有效降低黄萎病的发生,为棉花黄萎病的绿色防控提供了产品储备。

基于VFNet-Improved和Deep Sort的棉花黄萎病病情分级

棉花是全球最重要的经济作物之一,而黄萎病是世界主要棉花生产区的第一大病害,黄萎病病原菌通过感染棉花的根部使叶片萎蔫、褪色以致脱落,导致棉花质量和产量严重下降。国家标准将患黄萎病叶片划分为5个等级,传统检测方法主要依赖人工,存在主观、低效、重复性差等问题,因此提出一种以VFNet-Improved、Deep Sort和撞线匹配机制为主要算法框架的棉花黄萎病病情分级方法,实现在旋转视频输入情况下对患病叶片的数量统计和病情等级的划分。研究首先基于VFNet目标检测网络,融合多尺度训练、动态卷积等优化方法,实现对旋转视频中患病叶片的精准定位;然后采用Deep Sort跟踪器实现前后帧同一叶片的相互关联,并针对跟踪过程ID跳变问题设计了掩膜撞线匹配机制;最后使用OpenCV对经过掩膜线的叶片进行特征提取与患病分级的划分。试验结果表明,VFNet-Improved可以有效改善棉花患病叶片识别精度,mAP75达到0.906,较改进前VFNet模型提升了0.012,帧率FPS为12.9帧/s;Deep Sort跟踪器跟踪效果MOTA为0.835,对患病叶片数量统计结果R2、RMSE、MAE与MAPE分别为0.890、5.138、4.300和14.967%,与人工统计值具有较高一致性。本研究为棉花黄萎病病情精准、高效鉴定提供一种新的科学工具,对棉花抗病品种筛选和遗传机制解析具有重要意义。

类钙调磷酸酶B亚基蛋白GhCBL3-A01调控棉花黄萎病抗性的功能分析

【目的】解析类钙调磷酸酶B亚基蛋白(calcium B-like protein,CBL)基因GhCBL3-A01在棉花抗黄萎病反应中的功能,为棉花抗病育种提供基因资源。【方法】从棉花基因组数据库获得GhCBL3-A01基因的同源序列,进行生物信息学分析。采用实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测大丽轮枝菌(Verticillium dahliae)、茉莉酸甲酯(methyl jasminate,MeJA)和H2O2处理的棉株中GhCBL3-A01基因的表达量变化。利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术研究GhCBL3-A01在棉花抗黄萎病中的功能。通过检测活性氧积累以及相关基因的表达量,初步分析GhCBL3-A01的作用机制。【结果】陆地棉中GhCBL3-A01及其3个同源蛋白均含有3个CBL家族典型的EF-hand结构域。大丽轮枝菌、MeJA和H2O2处理后,GhCBL3-A01的表达量显著升高。VIGS沉默GhCBL3-A01导致病株率和病情指数显著降低,茎秆维管束褐变程度减轻,有病菌繁殖的茎段数量明显减少,增强了棉株对黄萎病的抗性。GhCBL3-A01基因沉默棉株叶片中活性氧积累增多,防御相关基因PR1、NPR1、PR4和PDF1.2以及茉莉酸信号通路关键基因AOS1、OPR3、MYC2和LOX2的表达量增加。【结论】GhCBL3-A01通过调控防御相关基因、茉莉酸信号通路基因的表达和活性氧积累负调控棉花对黄萎病的抗性,是提高棉花黄萎病抗性的候选基因。