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.

Bioinformatics Analysis of Antifungal Peptide 25a2 of Cotton Verticillium Wilt

Protein 25a2 is the antifungal peptide of cotton Verticillium wilt which was isolated from Bacillus amyloliquefaciens. The amino acid sequence of antifungal peptide 25a2 was analyzed using bioinformatics tools, and the characters of signal peptides, transmembrane topological structura, physicochemical signatures, protein domain, secondary and tertiary structure of protein were predicted. The results showed that 25a2 was a secreted protein, the sequence of which included a signal peptide in N end and a transmembrane domain in C end. The predicted secondary structure showed that the antibacterial peptide was mainly free random coils, belonging to mixed protein, three-dimensional model of 25a2 was a compact ball. These results showed that the most possible action mechanism of antifungal peptide 25a2 might be ＂carpet＂ model.

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.

Present Status, Problems and Prospects of Molecular Breeding of Cotton (Gossypium) with Resistance against Verticillium dahliae Kleb.

VerticiUium wilt is an important disease in cotton production, which seriously affects the production of cotton. The classification and pathogenesis of Yer-ticillium dahliae Kleb. , achievements in QTL mapping and molecular breeding were reviewed in the paper. Meanwhile, the current problems and future direction of resistance breeding against V. dahliae Kleb. were discussed so as to provide reference information for further research on resistance breeding against the pathogen.

外源添加L-脯氨酸对棉花黄萎病发生及其根际土壤微生物群落的影响

【目的】根系分泌物是植物与土壤微生物进行互作的信号媒介,对于植物病害发生和植株生长均具有重要调控功能。论文旨在明确棉花根系分泌物L-脯氨酸抵御棉花黄萎病发生的微生态机制,揭示L-脯氨酸介导的根际微生物与棉花黄萎病发生的互作关系,为构建生物防治土传病害的有益菌群提供新视角。【方法】通过温室盆栽试验,以外源添加不同浓度的L-脯氨酸(0、50、100、200和400 mmol·L^(-1))为试验处理,采用实时荧光定量PCR(real-time qPCR)和宏基因组测序技术分别测定不同处理的土壤中大丽轮枝菌(Verticillium dahliae)DNA拷贝数量和土壤微生物群落结构及功能,采用主成分分析比较不同处理的根际土壤微生物群落结构,利用冗余分析研究土壤养分因子与微生物群落结构的相关性,利用斯皮尔曼相关分析研究微生物群落结构功能代谢途径的相关性。【结果】与空白对照相比,低浓度(50 mmol·L^(-1))L-脯氨酸处理不能够减轻棉花黄萎病的发生,高浓度(100、200和400 mmol·L^(-1))L-脯氨酸处理的病情指数分别下降22.51%、60.23%和64.23%。qPCR结果表明,L-脯氨酸处理不能够显著降低土壤中大丽轮枝菌拷贝数量。宏基因组测序分析表明,L-脯氨酸处理后细菌多样性Shannon指数显著增加,真菌多样性Shannon指数呈下降趋势。在属水平上,L-脯氨酸处理后类诺卡氏菌属(Nocardioides)、溶杆菌属(Lysobacter)、节杆菌属(Arthrobacter)、Phycicoccus、假单胞菌属(Pseudomonas)和毛霉菌属(Mucor)的相对丰度呈上升趋势。线性判别分析表明,除浓度为100 mmol·L^(-1)的L-脯氨酸外,外源添加L-脯氨酸处理后根际土壤微生物KEGG通路的富集情况发生改变。冗余分析表明,细菌群落组成受pH、电导率、硝态氮、铵态氮和有机质显著影响,而真菌群落组成与铵态氮存在显著相关性。Spearman相关分析表明,细菌的KEGG代谢通路与pH、有机质、铵态氮含量呈负相关关系,而与电导率和硝态氮呈正相关关系;真菌的大部分KEGG代谢通路与土壤养分的相关性较差。【结论】外源添加适量L-脯氨酸通过改变土壤细菌群落结构和功能,增加有益微生物的相对丰度,从而影响棉花黄萎病的发生,但其不能够改变病原菌数量。同时,根际细菌群落组成和功能均与土壤养分存在相关性。

棉花黄萎病生防菌筛选鉴定及根瘤菌DG3-1对棉花生长的影响

本研究旨在发掘南疆棉花内生菌种质资源来防治棉花黄萎病,探究根瘤菌对棉花生长的影响。采用常规组织分离法分离棉花不同组织内生细菌;平板对峙法筛选拮抗棉花黄萎病菌的内生菌,并对其进行分子鉴定;通过盆栽试验研究根瘤菌对棉花生长的影响。结果显示,棉花不同组织内生细菌的数量具有一定差异,根部最多,其次是叶和茎;在分离获得的31株内生菌中,有11株对棉花黄萎病菌具有拮抗活性,抑菌率在35.99%~68.28%之间,经鉴定,9株为芽孢杆菌,2株为根瘤菌,其中,根瘤菌DG3-1对棉花黄萎病菌的抑制率为(56.63±1.82)%;盆栽试验结果表明,菌株DG3-1处理后棉株的地上鲜重、地上干重、地下鲜重、地下干重、叶绿素含量分别比对照增加2.12、2.09、1.05、0.75、1.75倍。菌株DG3-1对棉花黄萎病菌有较好的抑制作用,并且对棉花的生长有一定的促生作用,是防治棉花黄萎病的潜力菌株,可为微生物菌剂的研发提供菌种资源。

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。

Gh_D11G050000参与棉花黄萎病抗性的功能分析

【目的】克隆Gh_D11G050000基因,解析其在棉花黄萎病抗性反应中的功能和作用机制,为开展棉花抗病分子育种提供理论依据。【方法】利用生物信息学方法分析Gh_D11G050000基因的序列特征和系统进化关系。通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction, qRT-PCR)分析该基因的表达模式以及大丽轮枝菌(Verticillium dahliae)侵染后的表达量变化。利用病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术初步验证该基因的功能。通过转录组测序分析和相关基因的表达量测定初步解析其抗病机制。【结果】Gh_D11G050000与Gh_A11G049600和Gr_11G034620蛋白的亲缘关系最近。qRT-PCR分析发现,Gh_D11G050000在棉花根部的表达量最高;该基因的表达量在大丽轮枝菌侵染后显著增加。Gh_D11G050000沉默植株对大丽轮枝菌的抗性减弱,具体表现为茎秆维管束褐变加重,有病菌繁殖的茎段数量明显增加,病株率和病情指数显著升高。转录组及qRT-PCR分析表明,Gh_D11G050000沉默棉株中茉莉酸、乙烯信号通路和木质素合成途径中相关基因的转录水平降低。【结论】Gh_D11G050000通过影响木质素合成基因、茉莉酸和乙烯信号通路相关基因的表达正调控棉花对黄萎病的抗性。

棉花抗黄萎病相关基因GhERF020功能的初步分析

黄萎病(Verticillium wilt)是影响我国棉花产量和品质的重要病害,其病原菌是大丽轮枝菌(Verticillium dahliae)。AP2/ERF类转录因子在植物生长发育和胁迫适应性响应中的作用日益突显。为挖掘棉花抗黄萎病相关基因并探究其生物学功能,利用拟南芥抗大丽轮枝菌相关基因AT1G22810.1,同源比对获得棉花抗黄萎病候选基因GhERF020(XM_016842745.1)。生物信息学分析结果表明,GhERF020开放阅读框的长度为441 bp,编码147个氨基酸;其蛋白含有1个保守的AP2结构域,相对分子质量为16.16 kD,无跨膜结构,属于亲水蛋白;预测其启动子区域含有水杨酸、茉莉酸甲酯和脱落酸等响应元件。基因表达模式分析发现,GhERF020受大丽轮枝菌侵染诱导表达。亚细胞定位分析表明,GhERF020蛋白定位于细胞核中。利用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术下调GhERF020表达后,棉花植株对大丽轮枝菌的敏感性显著增强,病情指数和病原菌生物量明显升高。由此表明,GhERF020是棉花抗黄萎病的正向调控因子。

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)。

生物药剂复配对棉花黄萎病防治及生长发育的影响

【目的】研究生物药剂复配对棉花黄萎病防治效果及生长发育的影响,为棉花植株健康生长提供理论依据。【方法】于2022年进行大田试验,选用棉花品种为新陆中42号。药剂以80%乙蒜素乳油药液、亚磷酸钾、谷胱甘肽为基础,分别为复配抗病毒专用型助剂(T_(1))、复合微生物菌肥(T_(2))、15%噁霉灵(T_(3))、5%辛菌胺醋酸盐(T_(4))、25%吡唑醚菌酯(T_(5)),分析不同药剂对棉花黄萎病防治效果,农艺性状及产量的影响。【结果】各处理对棉花病害防治效果显著,对棉花生长表现出一定的促进作用,增加棉花株高、果枝数和蕾铃数。其中T_(2)处理较T_(1)和T_(3)处理对棉花黄萎病防治效果提高19.99%、23.34%,T_(1)、T_(2)处理较CK处理铃数增加1.8个、2.67个。T_(2)处理的籽棉产量为6104.86 kg/hm^(2),较其他处理产量最高并差异显著。【结论】滴施复合微生物菌肥+80%乙蒜素乳油+亚磷酸钾+谷胱甘肽(T_(2))可提高黄萎病防治效果,增加蕾数铃数和单株结铃数,从而显著增加棉花产量。