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.

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.

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.

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.

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.

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.

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.

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.

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.

Field Application of Non-Pathogenic <i>Verticillium dahliae</i>Genotypes for Regulation of Wilt in Strawberry Plants

Verticillium dahliae induced wilt disease in strawberry can cause severe yield losses and thus lead to inevitable negative economic impacts. Inoculation of plants with non-pathogenic strains of Verticillium was conducted as a biologic control agent (BCA) according to the concept that preoccupation of the ecologic niche rendered strawberry plants immune to infection with soil-borne pathogenic Verticillium. This concept was tested for economic viability in a field trial under commercial conditions. Results were reported for 2 years of field trials under practice conditions in two locations in Brandenburg, Germany. Inoculation was shown to have a positive effect of 20% of plants, while 30% of plants remain unaffected and of equally high vitality. However, 50%-0% of plants were impacted negatively, showing severe wilt symptoms up to total loss. The characteristic progression of wilt symptoms suggested an infestation caused by Phytophtora sp. and other pathogens. Further results showed that the main factor of the side effects was caused by different qualities of plant material in interaction to the inoculation with the BCA and only to a minor extent depended on pre-infestation of soils. We conclude that specific conditions, such as certified plant material or soil analysis for other pathogens besides Verticillium, avoided these side-effects relevant for commercial farming.

QTL Analysis of Fiber Yield and Quality and Resistance to Verticillium Wilt Using Gossypium hirsutum and G.barbadense Advanced Backcross Populations

To introgress elite QTL alleles of Gossypium barbadense L.for fiber yield and quality and resistance to Verticillium wilt into G.hirsutum L.,enlarge the genetic base of G.hirsutum,and

Control of verticillium wilt in Cotinus coggygria

We analyzed the inhibition rates of eight kinds of fungicides to the pathogen of verticillium wilt of smoke tree （Cotinus coggygria） by measuring mycelium growth. Results show that four fungicides （Weijunjing, thiophanate-methyl 70% WP （wettable powder）, carbendazim 50% WP and Junxianwei） have the best antifungal effects. Three fungicides, Weijunjing, carbendazim 50% WP and Junxianwei, were selected to determine the control of verticillium wilt in potted smoke tree seedlings. We found that the relative efficacy of Weijunjing was up to 74%. Based on the results, Weijunjing and carbendazim 50% WP were prepared for field trials. The results indicate that the relative efficacy of Weijunjing （300 times diluted） and carbendazim 50% WP （400 times diluted） were 66.2% and 48.9% in plot 1 and 26.4% and 31.8% in plot 2 after soil disinfection, and were 224% and 61.8% in plot 3 without soil disinfection in 2007. The relative efficacy of Weijunjing and carbendazim 50% WP were 49.6% and 45.4% in plot 2 in 2008.

Field Efficiency Trial of Microbial Fertilizer against Eggplant Verticillium Wilt

The control effect of microbial fertilizer on eggplant verticillium wilt was studied through investigation of disease index and diseased plant rate. The reiults showed that the disease index and diseased plant rate of eggplant verticillium wilt were the lowest in the plots with substrate treatment and root-irrigation treatnent. The control effects in the plots with substrate treatment were higher than those in the plots with only root irrigation treatment. Moreover, higher concentration of microbial fertilizer resulted in better control effect and higher yield. Thus, the microbial fertilizer had a broad application prospect.

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

GbAt11 gene cloned from Gossypium barbadense mediates resistance to Verticillium wilt in Gossypium hirsutum

Background:Gossypium hirsutum is highly susceptible to Verticillium wilt,and once infected Verticillium wilt,its yield is greatly reduced.But G.barbadense is highly resistant to Verticillium wilt.It is possible that transferring some disease-resistant genes from G.barbadense to G.hirsutum may contribute to G.hirsutum resistance to Verticillium wilt.Result:Here,we described a new gene in G.barbadense encoding AXMN Toxin Induced Protein-11,Gb At11,which is specifically induced by Verticillium dahliae in G.barbadense and enhances Verticillium wilt resistance in G.hirsutum.Overexpression in G.hirsutum not only significantly improves resistance to Verticillium wilt,but also increases the boll number per plant.Transcriptome analysis and real-time polymerase chain reaction showed that Gb At11 overexpression can simultaneously activate FLS2,BAK1 and other genes,which are involved in ETI and PTI pathways in G.hirsutum.Conclusion:These data suggest that Gb At11 plays a very important role in resistance to Verticillium wilt in cotton.And it is significant for improving resistance to Verticillium wilt and breeding high-yield cotton cultivars.

Transcriptome Analysis of a Wild Eggplant Germplasm M239 in Response to Verticillium dahliae Infection

In this study,wild eggplant germplasm No.M239,which is highly susceptible to Verticillium wilt,was used as the experimental material.The physiological and biochemical indices(SOD,PAL,MDA and soluble protein)of M239 roots were measured at different times(0,12,24,36,48,60 and 72 h)post inoculation with Verticillium dahliae,and the key time points for the M239 response to Verticillium wilt infection were screened.Then,RNA-Seq technology was used to screen the differentially expressed genes(DEGs)in M239 roots at 0,12 and 48 h post-inoculation(hpi).The transcriptional results of M239 were also compared with those resistance genes from some reported wild relative Solanum species(S.sisymbriifolium and S.aculeatissimum).Then some DEGs were chosen for validation by qRT–PCR.The results showed that 12 and 48 hpi were the turning points in the changes in all physiological and biochemical indices.A total of 6,783 DEGs were identified by RNA-Seq,including 6,141 DEGs(3,046 upregulated and 3,095 downregulated)at the M_12 h vs.M_0 h,1,903 DEGs(792 upregulated and 1,111 downregulated)at M_48 h vs.M_12 h,and 1,261 DEGs that appeared simultaneously in both stages.KEGG enrichment analysis showed that there were 5 metabolic pathways enriched from DEGs,which were mostly related to primary metabolism,such as glycolysis,amino acid and ribosome biogenesis.Compared with the NCBI non-redundant protein(NR)database,one Ve2 homologous gene and 8 PR protein-related genes were screened.Transcription factor analysis showed that there were a large number of DEGs,such as MYB,AP2-EREBP,bHLH,NAC and Orphans in the two stages.Compared with the reported Verticillium wilt-resistant wild eggplant species,it was found that there were fewer genes and enriched metabolic pathways in the M239 response to Verticillium wilt infection,and it also lacked the response of some known key resistance genes.These results proved that the above resistance genes and metabolic pathways played a key role in the wild eggplant response to V.dahliae infection.

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。

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.

八达岭林场黄栌枯萎病防效研究

【目的】研究不同药剂组合和施药技术对八达岭林场黄栌枯萎病林间控制效果,探讨黄栌枯萎病有效防控措施,旨在保障北京地区黄栌健康和红叶景观安全。【方法】选择枯草芽孢杆菌、50%嘧菌酯、156 g/L丙环唑、50%多菌灵和45%咪鲜胺等5种药剂,采用灌根、树干注射和两者相结合的施药方式,共设置12个处理,对八达岭林场黄栌枯萎病开展林间防治试验。【结果】通过比较各处理在2021—2022年间黄栌枯萎病病情指数,得出当年防治效果最好的施药组合是灌根丙环唑结合树干注射多菌灵与嘧菌酯复配,校正病情指数为5,防治效果达到88%。并且,该组合中有26.67%植株保持健康,健康植株数量最多。第2年5月病情指数最低的施药组合是灌根枯草芽孢杆菌结合树干注射多菌灵与嘧菌酯复配,表现出较好的治疗效果,健康植株数量最多,且往年重度发病样树均转为无病或轻度发病,病情指数仅有3.33。其他施药方式和药剂组合均在不同程度上缓解黄栌枯萎病的发生,但是不同处理的防治效果统计学上存在显著差异(P <0.05)。【结论】本研究结果表明灌根丙环唑和树干注射多菌灵与嘧菌酯复配组合防治效果最佳,灌根枯草芽孢杆菌结合树干注射多菌灵与嘧菌酯复配组合在感病治疗及防治效果方面均表现良好,两者均可作为黄栌枯萎病的防治方案。

陆地棉抗黄萎病基因GhENODL6互作蛋白筛选

研究发现陆地棉ENODL6基因具有抗黄萎病功能。为进一步揭示其在棉花抗黄萎病过程中发挥的作用,通过Nimble Cloning技术将GhENODL6插入酵母表达载体pNC-GBKT7,构建了重组质粒pNC-GBKT7-ENODL6。重组质粒转入酵母菌Y2HGold后可在DDO培养基上正常生长,但不能生长于QDO/X/A培养基,表明GhENODL6蛋白对酵母宿主无毒害作用,没有自激活活性。将携带pNC-GBKT7-ENODL6诱饵载体的Y2HGold酵母菌与cDNA文库进行杂交筛选,结果获得一个能够显示蓝色的菌落。通过PCR扩增,在蓝色酵母菌中获得一段526 bp的非载体插入片段,与陆地棉基因组内基因WRKY47序列高度一致。通过同源扩增,从陆地棉中克隆到WRKY47开放读码框,全长1 587 bp,编码528个氨基酸残基。再次利用酵母双杂交技术确认了WRKY47与GhENODL6之间存在互作关系。综上,构建了重组载体pNC-GBKT7-ENODL6,并鉴定到与GhENODL6互作的蛋白WRKY47。