Comparative Assessment of Genetic Diversity of Peanut (Arachis hypogaea L.)Genotypes with Various Levels of Resistance to Bacterial Wilt Through SSR and AFLP Analyses

Bacterial wilt （BW） caused by Ralstonia solanacearum is an important constraint to peanut （Arachis hypogaea L.） production in several Asian and African countries, and planting BW-resistant cultivars is the most feasible method for controlling the disease. Although several BW-resistant peanut germplasm accessions have been identified, the genetic diversity among these has not been properly investigated, which has impeded efficient utilization. In this study, the genetic relationships of 31 peanut genotypes with various levels of resistance to BW were assessed based on SSR and AFLP analyses. Twenty-nine of 78 SSR primers and 32 of 126 AFLP primer combinations employed in this study were polymorphic amongst the peanut genotypes tested. The SSR primers amplified 91 polymorphic loci in total with an average of 3.14 alleles per primer, and the AFLP primers amplified 72 polymorphic loci in total with an average of 2.25 alleles per primer. Four SSR primers （14H06, 7G02, 3A8, 16C6） and one AFLP primer （P1M62） were found to be most efficient in detecting diversity. The genetic distance between pairs of genotypes ranged from 0.12 to 0.94 with an average of 0.53 in the SSR data and from 0.06 to 0.57 with an average of 0.25 in the AFLP data. The SSR-based estimates of the genetic distance were generally larger than that based on the AFLP data. The genotypes belonging to subsp, fastigiata possessed wider diversity than that of subsp, hypogaea. The clustering of genotypes based on the SSR and AFLP data were similar but the SSR clustering was more consistent with morphological classification ofA. hypogaea. Optimum diverse genotypes of both subsp, hypogaea and subsp.fastigiata can be recommended based on this analysis for developing mapping populations and breeding for high yielding and resistant cultivars.

Effects of Seedling-cake Integrated with Arbuscular Mycorrhizal Fungi on Resistance of Bacterial Wilt in Pepper (Capsicum annuum L.)

[Objective] The aim was to investigated the effects of seedling-cake with AMF on resistance of pepper （Capsicum annuum L.） to bacterial wilt. [Methods] A pot experiment was conducted to evaluate the dynamics of mycorrhizal colonization, disease index, morbidity rate, control effect and several agronomic traits of mycorrhizal seedlings after inoculation with RS in two kinds of seeding-cakes with AMF. [Results] Two kinds of seedling-cakes formed steadily mycorrhizal colonization after inoculation, which enhanced disease-resistance and decreased morbidity rate and disease index in different degrees. What’s more, the performance of self-made seedling-cakes was obviously better than that of commercial seedling-cakes. [Conclusions] Self-made seedling-cakes with AMF have superior performance on bacterial wilt resistance of pepper, which should be made use of in other crops extensively.

Effect of Fertilization on Tomato Bacterial Wilt Biocontrolling and Soil Health Restoration Using FAME Analysis

The effect of applying biological organic fertilizer(BOF)on bacterial wilt incidence of tomato and soil microbial community under continuous cropping was studied. The results showed that all the tomatoes were infected by bacterial wilt in the control. The infection rates of tomatoes in the treatments with un-composted BOF and decomposed BOF were 55 and 50% respectively. Fatty acid methyl esters(FAME)analysis indicated soil microbial community changed sensitively after applying BOF. Soil FAME total content, relative fungi content and ratio of fungi to bacteria were significantly increased in both BOF treatments. The soil odd-number fatty acid proportion changed after applying BOF, aC15 : 0, iC17 : 0 decreased, while cyC17 : 0 increased in soil odd-number fatty acid proportion. BOF application would strengthen soil health and disease suppression. The content of C16 : 1 11c in soil microbial community was obviously increased after decomposed BOF application. It indicated that the growth of AM fungi could be enhanced with decomposed BOF application. FAME microbial biomarkers could be used for an indicator of soil health and disease suppression. Odd-number fatty acid proportion was a sensitive indicator of the effect of applying un-composted and decomposed BOF respectively on soil health regulating.

Control Effect of Different Microbial Agents on Tobacco Bacterial Wilt

[ Objectives] The paper was to study effects of different microbial agents on occurrence of tobacco bacterial wilt. [ Methods I Antagonistic strains against Ralstonia solanacearum were screened from in situ soil, and prepared into four different combinations. The number of soil microorganisms and incidence of bacterial wilt of four combinations were observed. [ Results] The incidence rate of bacterial wilt treated by microbial agent OR-1 was significantly lower than that in control, and the control efficacy against bacterial wilt reached 67%. [ Conclusions] Application of microbial agent OR-1 could effectively reduce the incidence rate and disease index of bacterial wilt, thus improving the quality of tobacco.

Effect of Integrated Use of Inorganic Fertilizer and Organic Manures on Bacterial Wilt Incidence (BWI) and Tuber Yield in Potato Production Systems on Hill Slopes of Central Kenya

Bacterial wilt (BW) caused by Ralstonia solanacearum is one of the most damaging diseases of potato (Solanum tuberosum L.) in Kenya and worldwide. In Kenya, potato tuber yield losses due to BW infection are estimated at 50 ~ 100%. Low soil fertility is also one of the most important constraints limiting potato production in central Kenya highlands. Farmers tackle this problem through use of inorganic fertilisers and organic manures, both of which amend the soil environment to influence bacterial wilt development. Undecomposed organic manures can also introduce the pathogen into a clean field. Between short rains 1999 and 2000, 10 on-farm extension-researcher- farmer-designed and farmer-managed trials were done at Kianjuki catchment in Embu District. The objective was to use farmers?participatory research approach and select the most suitable organic and inorganic fertiliser combination(s) with lowest BWI and acceptable usable tuber yields, and also to demonstrate use of some components of integrated disease management methods in reduction of disease incidence and spread. Seven treatments were proposed, presented to the farmers for discussion and the most relevant four were selected for evaluation. A newly released potato variety 慉sante?was planted during the short rains 1999 and long rains 2000. BWI didn抰 result in significant differences between treatments but the tuber yields were significantly different in short rains of 1999 and 2000. During short rains 2000, both BWI and tuber yields and unusable tubers differed significantly between treatments. The results confirmed that well- decomposed manures or manures from pathogen-free areas can be used in combination with inorganic fertilisers to improve soil fertility and potato tuber yields in smallholder farms without influencing BWI. Use of certified seed tubers in pathogen free field and following recommended field sanitation measures resulted in apparently bacterial wilt free crop. Considering the high cost of inorganic fertiliser and its negative effects on the environment, reduced usage at half the recommended rates combined with half rates of FYM is feasible option friendly to the farmer, soil and environment. The interviewed farmers also ranked the option as the most appropriate combination for soil fertility improvement for potato production in smallholder farms.

Effects of Bio-organic Fertilizer with Antagonistic Bacteria against Tobacco Bacterial Wilt on Soil Microbial Communities and Disease Resistance of Tobacco

In this study, the effects of bio-organic fertilizer with antagonistic bacteria against tobacco bacterial wilt on soil microbial communities and disease resist- ance of tobacco were investigated by field experiment. The results showed that the incidence of tobacco bacterial wilt in bio-organic fertilizer treatments （T3 and T4） decreased remarkably among four treatments in the field. Compared with the local conventional fertilization group, the incidence of tobacco bacterial wilt was re- duced by 21.9% and 25.0% in T3 and T4, respectively ; the yield of flue-cured tobacco was improved by 5.7% and 5.3%, respectively ; the proportion of mid- high grade tobacco leaves increased by 2.3% and 2.6%, respectively. After application of bio-organie fertilizer with antagonistic bacteria against tobacco bacterial wilt, rhizosphere soil microbial communities exhibited vast amount and abundant species ; the amount of rhizosphere soil bacteria of infected tobacco plants was im- proved by 218.5% with fewer species. It could be concluded that the application of bio-organic fertilizer with antagonistic bacteria against tobacco bacterial wilt could improve the ecological environment of tobacco field, inhibit the growth of pathogenic bacteria, decrease the incidence of tobacco bacterial wilt, and enhance the quality of flue-cured tobacco. This study laid the foundation for further ecological prevention and control of soil-borne diseases of tobacco.

Integrated Biological Control of Tobacco Bacterial Wilt (<i>Ralstonia solanacearum</i>) and Its Effect on Rhizosphere Microbial Community

Biological control agents and soil amendments have been applied to control tobacco bacterial wilt, but the mechanism is not well-known. In the present study, a field experiment was performed to investigate the soil physicochemical properties, disease index (DI) and disease incidence of tobacco bacterial wilt, and rhizosphere microbial community. The results show that the control efficacy of single application of antagonistic bacteria and calcium cyanamide was 46.43% and 51.92%, respectively. While the combined control efficacy of antagonistic bacteria and calcium cyanamide was 65.79%. Besides, the combined application of antagonistic bacteria and calcium cyanamide could increase soil pH, total N alkaline N, and exchangeable Ca, which were negatively associated with the microbial diversity, soil-borne pathogenic microorganisms, and incidence of tobacco bacterial wilt. Additionally, the combination of antagonistic bacteria and calcium cyanamide can improve the proportion of some antagonistic microbial species, and these antagonistic microbial species were inversely associated with the DI of tobacco bacterial wilt. In conclusion: The integrated measure may influence soil microbial communities through enhancing soil physicochemical properties and rebuild healthy soil microbial community structure to mitigate tobacco bacterial wilt. The current study presented valuable insights into the mechanisms enhancing soil health in the integrated measure.

Screening, Identification and Biocontrol Effect of Antagonistic Streptomyces Strain Tra69 against Tobacco Bacterial Wilt

[Objective] The paper was to screen the biocontrol agents with good control effects against tobacco bacterial wilt. [Method] The strains with antagonistic activity against tobacco bacterial wilt were isolated from 36 copies of rhizosphere soil sample. The strain with best inhibition effect was identified, and carried out pot test and growth-promoting experiment. [Result] The strain with best inhibtion effect was Tra69, which was identified to be Streptomyces flavogriseus. The fermentation liquid of Tra69 had good control effect against Ralstonia solanacarum, and also had good growth-promoting effect on tobacco. [Conclusion] Tra69 was the biocontrol agent with excellent control effect against R. solanacarum, which could be further developed and used in biological control against tobacco diseases.

Threshold of Soil pH for Occurrence of Tobacco Bacterial Wilt in Henan Province

[Objective]The paper was to study the relationship between spatial distribution characteristics of soil p H and bacterial wilt in tobacco-growing area in Henan Province.[Method]The 21 tobacco-growing fields infected by bacterial wilt and 91 tobacco-growing fields with no cases were selected from Henan Province,and the pH of fields was determined.[Result]Bacterial wilt mainly occurred in the tobacco-growing fields in Luoshan,Pingqiao,Queshan,Biyang,Suiping,and Xiping,and the soil pH mainly ranged from 5.5 to 6.5(accounted for 84.75%of total surveyed tobacco fields);bacterial wilt had not been found in tobacco-growing fields in Tanghe,Zhengyang,Shangcai,Sheqi,Wuyang and Chenqu,and the soil pH mainly ranged from 6.5 to 7.0(accounted for 81.25%of total surveyed tobacco fields).There were significant differences in soil pH between the fields with and without bacterial wilt,and soil pH 6.5 could be regarded as the threshold for the occurrence of tobacco bacterial wilt in Henan Province.There was extremely significantly positive linear correlation between soil p H and latitude(y=0.7375 x-17.799,R2=0.386,P<0.01).The geographical boundary of soil pH 6.5 was roughly 33°N,and bacterial wilt was less likely to occur in the north tobacco-growing areas of the boundary.[Conclusion]The study will provide a scientific basis for the prevention and control of tobacco bacterial wilt in Henan.

Genome-wide investigation of defensin genes in peanut(Arachis hypogaea L.)reveals AhDef2.2 conferring resistance to bacterial wilt

Peanut(Arachis hypogea L.)bacterial wilt(BW)is a devastating disease caused by Ralstonia solanacearum that results in severe yield and quality losses.Plant defensins are short cysteine-rich peptides with antimicrobial activity.The role of defensin genes(Ah Def)in peanut is unclear.A genome-wide investigation of Ah Def genes was undertaken,and 12 identified Ah Def genes were classified into two groups containing the gamma-thionin domain formed by four disulfide pairs:Cys1-Cys8,Cys2-Cys5,Cys3-Cys6,and Cys4-Cys7.In silico analysis revealed that Ah Def genes showed highly conserved architectural features and contained cis-elements associated with phytohormone signaling and defense responses.A highly resistant cultivar,H108(R)and a susceptible accession,H107(S)were tested by R.solanacearum inoculation.H108(R)showed fewer symptoms than H107(S)owing to inhibition of bacterial reproduction and spread in the vascular bundles of roots and stems.In a transcriptomic expression profile,Ah Def genes,particularly Ah Def1.6 and Ah Def2.2,were up-regulated in H108(R)compared with H107(S)under R.solanacearum infection and phytohormone treatment.Subcellular localization showed that the Ah Def1.6 and Ah Def2.2 proteins were both expressed specifically on the plasma membrane.Overexpression of protein fusion Ah Def2.2-YFP in Nicotiana benthamiana and peanut leaves increased resistance to R.solanacearum,suggesting its role in response to BW infection.Ah Def2.2 may be valuable for peanut resistance breeding.

Effects of Integrated Technology of Medicine and Fertilizer on Bacterial Wilt of Tobacco

In order to validate the control effects of integrated technology of medicine and fertilizer on tobacco bacterial wilt,field experiments were conducted by using youmeili and tetracycline as the test agents.The results obtained were listed as follows:(1)The incidence rate of bacterial wilt ascended more quickly after July 10 th,the incidence of CK reached 78.30%on July 25 th.(2)The control effect of“youmeili 3 g+tetramycin 200 times liquid”reached 69.47%,significantly higher than other treatments.(3)The output value of“Youmeil 3 g+tetramycin 200 times liquid”served as 1635.66 yuan/667m^2,with the highest economic character in all treatments.In a word,integrated technology of medicine and fertilizer was a feasible approach to control tobacco bacterial wilt.

Analysis of Meteorological Epidemic Factors and Soil Improvement Controlling Technology of Tobacco Bacterial Wilt (Ralstonia solanacarum)

[Objective] The paper was to analyze the meteorological epidemic factors for occurrence and prevalence of tobacco bacterial wilt （ Ralstonia solanaca- rum）, and to study control effects of different soil conditioners on the bacterial disease in Gacligongshan demonstration area of green, ecological, high quality tobac- co leaf production. [Method] The plots attacked by tobacco bacterial wilt over the years were selected and the incidence of the disease was periodically surveyed in tobacco growth period in 2012, 2103 and 2014, respectively. 10 d Effective accumulated temperature and rainfall were counted according to the meteorological data, and the relationship between meteorological factors and disease index was analyzed. The control effects of three kinds of soil conditioners ＂Zhuanggenfeng＂, refined fulvic acid and lime on tobacco bacterial wilt were tested. [ Result] The analysis results of meteorological factors showed that 10 d effective accumulated temperature and rainfall were positively correlated to disease index. The variation curve of 10 d effective accumulated temperature and rainfall reflected the change trend of disease index. The pH values were increased by 0.57, 0.50 and 0.72 respectively after applying ＂Zhuanggenfeng＂, refined fulvic acid and lime. The aver- age control effects on tobacco bacterial wilt were 60.74% -62. 18%, 53.05% -59.53%, and 48.59% -58.53%, respectively. [ Conclusion] 10 d Effective accumulated temperature and rainfall could be used as important reference for disease forecasting and controUing. The usage of soil conditioner has a certain preven- tion and control effect on tobacco bacterial wilt disease by forming soil conditions conducive to flue-cured tobacco growth but adverse to disease survival, which is an effective auxiliary method against the disease.

Improvement of Bio-Efficacy to Reduce Bacterial Wilt Complex Disease in Tomatoes through Bacillus amyloliquefaciens and Pseudomonas fluorescens under Field Conditions

Bacterial wilt complex disease of tomato(Solanum lycopersicum L.)was incited jointly by bacterial wilt pathogen Ralstonia solanacearum and Meloidogyne incognita worldwide.Bio-efficacy of bacterial antagonists i.e.B.amyloliquefaciens DSBA-11 and P.fluorescens DTPF-3 was studied against the wilt disease complex in tomato at National Phytotran facility Indian Agricultural Research Institute(IARI),New Delhi,at 26±2°C.Minimum wilt disease incidence(26.00%)with the highest bio-control efficacy(64.15%),less juvenile population(19.33 J2/g of soil)of M.incognita was recorded in the combined application of DTPF-3+DSBA-11 after 30 d of inoculation under glasshouse conditions.In a field study,minimum bacterial wilt disease incidences 19.0%and 20.4%were recorded in the bleaching powder treatment followed by mixed application of DSBA-11+DTPF-3,19.6%and 21.2%wilt incidence in 2014 and 2015 respectively.However,a reduction of root-knot gall index was recorded a maximum of 59.76%and 69.62%in DSBA-11+DTPF-3 treated plants followed by 54.88%and 60.13%over control in DTPF-3 treatment in 2014 and 2015 respectively.The yield of tomato fruit was increased over control by 17.48%and 16.97%in 2014 and 2015 respectively under field conditions.A combination of P.fluorescens DTPF-3+B.amyloliquefaciens DSBA-11 suppressed bacterial wilt and root-knot diseases and also increased the yield of the tomato fruit significantly(p<0.05)under field conditions.

Seedling Petri-dish inoculation method:A robust,easy-to-use and reliable assay for studying plant-Ralstonia solanacearum interactions

Ralstonia solanacearum causes a lethal bacterial wilt disease in many crops,leading to huge losses in crop production every year.Understanding of plant-R.solanacearum interactions will aid to develop efficient strategies to control the disease.As a soilborne pathogen,R.solanacearum naturally infects plants via roots.A huge limitation in studying plant-R.solanacearum interactions is the large variation of R.solanacearum infection assay due to the variable soil conditions and uneven inoculum exposure.Here,we developed a robust and reliable Petri-dish inoculation method which allows consistent and stable infection in young plant seedlings.This method is easy to use,takes about only 10 days from seed germination to the completion of inoculation assay,and requires less inoculum of bacteria as well as growth chamber space.We proved the efficacy of the seedling Petri-dish inoculation method by analyzing plant defense primed by molecular patterns,resistance of defense-related plant mutants,and virulence of R.solanacearum mutants.Furthermore,we demonstrated that the seedling Petri-dish inoculation method can be applied to other host plants such as tobacco and has great potential for high-throughput screening of resistant plant germplasms to bacterial wilt in the future.

A bacterial effector protein uncovers a plant metabolic pathway involved in tolerance to bacterial wilt disease

Bacterial wilt caused by the soil-borne plant pathogen Ralstonia solanacearum is a devastating disease worldwide.Upon plant colonization,R.solanacearum replicates massively,causing plant wilting and death;collapsed infected tissues then serve as a source of inoculum.In this work,we show that the plant metabolic pathway mediated by pyruvate decarboxylases(PDCs)contributes to plant tolerance to bacterial wilt disease.Arabidopsis and tomato plants resp ond to R.solanacearum infection by in creasing PDC activity,and plants with deficient PDC activity are more susceptible to bacterial wilt.Treatment with either pyruvic acid or acetic acid(substrate and product of the PDC pathway,respectively)enhances plant tolerance to bacterial wilt disease.An effector protein secreted by R.solanacearum,RipAK,interacts with PDCs and inhibits their oligomerization and enzymatic activity.Collectively,our work reveals a metabolic pathway involved in plant resistance to biotic and abiotic stresses,and a bacterial virulence strategy to promote disease and the completion of the pathogenic life cycle.

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt(caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying.However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected,suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

Silicon impacts on soil microflora under Ralstonia Solanacearum inoculation

Silicon(Si) can increase plant resistance against bacterial wilt caused by Ralstonia solanacearum and enhance plant immune response. However, whether Si alleviates soil-borne disease stress through altering soil microbial community component and diversity is not clear. In this study, effects of Si application under R. solanacearum inoculation with or without plant on soil bacterial and fungal communities were investigated through high-throughput pyrosequencing technique. The results showed that Si addition significantly reduced bacterial wilt incidence. However, Si did not reduce the amount of R. solanacearum in rhizosphere soil. Principal components analysis showed that soil microbial community composition was strongly influenced by Si addition. Total 63.7% bacterial operational taxonomic units(OTUs) and 43.8% fungal OTUs were regulated by Si addition regardless of the presence of tomato plants, indicating the independent effects of Si on soil microbial community. Si-added soil harbored a lower abundance of Fusarium, Pseudomonas, and Faecalibacterium. Our finding further demonstrated that exogenous Si could significantly influence soil microbial community component, and this may provide additional insight into the mechanism of Si-enhanced plant resistance against soil-borne pathogens.

Cloning of Proteinase Inhibitor Gene StPI in Diploid Potato and Its Expression Analysis

A full-length cDNA of proteinase inhibitor gene with completed open reading frame of 116 amino acids was cloned from Ralstonia solanacearum （Rs） resistant potato leaves using the rapid amplification of cDNA ends （RACE） method and designated as StPI. BLAST search against NCBI showed that the StPI gene shared 89% identity with potato proteinase inhibitor I precursor in nucleotide and 74% in amino acid. Analysis of semi-quantitative RT-PCR indicated that this gene was induced by Rs as well as up-regulated by jasmonic acid （JA）. The StPI gene expression reached the highest level during 6-12 h post Rs-inoculation or JA-treatment, and then leveled off. Moreover, this gene was strongly induced by JA and its mRNA accumulation increased more quickly than that of Rs-inoculation. The StPI gene may play a role in potato resistance against Rs. The induction of StPI by Rs invasion may have a similar signal transduction pathway with JA treatment.

The relative importance of soil moisture in predicting bacterial wilt disease occurrence

Soil-borne plant diseases cause major economic losses globally.This is partly because their epidemiology is difficult to predict in agricultural fields,where multiple environmental factors could determine disease outcomes.Here we used a combination of field sampling and direct experimentation to identify key abiotic and biotic soil properties that can predict the occurrence of bacterial wilt caused by pathogenic Ralstonia solanacearum.By analyzing 139 tomato rhizosphere soils samples isolated from six provinces in China,we first show a clear link between soil properties,pathogen density and plant health.Specifically,disease outcomes were positively associated with soil moisture,bacterial abundance and bacterial community composition.Based on soil properties alone,random forest machine learning algorithm could predict disease outcomes correctly in 75%of cases with soil moisture being the most significant predictor.The importance of soil moisture was validated causally in a controlled greenhouse experiment,where the highest disease incidence was observed at 60%of maximum water holding capacity.Together,our results show that local soil properties can predict disease occurrence across a wider agricultural landscape,and that management of soil moisture could potentially offer a straightforward method for reducing crop losses to R.solanacearum.

Effect of Soil Conditioner on the Microbial Community in the Rhizosphere Soil of Tobacco

The effects of biochar as a soil conditioner on microbial communities were studied. The results showed that, at all the growth stages of tobacco, the quantity of microorganisms in the rhizosphere soil added with the soil conditioner was higher than control （CK）. The results of metagenome detection indicated that the diversity of microbial communities in soil added with the soil conditioner was also higher than the CK, and the rate of tobacco bacterial wilt and the disease index decreased by 27.6% and 34.6%, respectively, compared with the CK. In conclusion, the soil con- ditioner could reduce the soil-borne disease by improving micro-ecological environ- ment and diversity of microbial communities in rhizosphere soil to maintain the soil balance.