H+-ATPase as a biochemical marker for early detection of root (wilt) disease in coconut palms (Cocos nucifera L).

H+-ATPase activity in leaves and roots of coconut palms growing in 'root wilt disease-prevalent areas' was compared with that of coconut palms growing in 'disease-free areas'. The activity was found to be significantly less in the leaves and roots of palms in the disease-prevalent zone as compared to that in disease-free zone. Histochemical examination of the leaves showed results that corroborated the biochemical findings. The possible application of H+-ATPase activity as a marker for the early detection of wilt disease in coconut palms is suggested.     

Nested and TaqMan® probe based quantitative PCR for the diagnosis of Ca. Phytoplasma in coconut palms

The root (wilt) disease caused by phytoplasma (Ca. Phytoplasma) is one of the major and destructive occurs in coconut gardens of Southern India. As this organism could not be cultured in vitro, the early detection in the palm is very much challenging. Hence, proper early diagnosis and inoculum assessment relay mostly on the molecular techniques namely nested and quantitative PCR (qPCR). So, the present study qPCR assay conjugated with TaqMan^® probe was developed which is a rapid, sensitive method to detect the phytoplasma. For the study, samples from different parts of infected coconut palms viz., spindle leaflets, roots and the insect vector—leaf hopper (Proutista moesta) were collected and assessed by targeting 16S rRNA gene. Further, nested PCR has been carried out using p1/p7 and fU5/rU3 primers and resulted in the amplification product size of 890 bp. From this amplified product, specifically a target of 69 bp from the 16S rRNA gene region has been detected through primers conjugated with Taqman probe in a step one instrument. The results indicated that the concentration of phytoplasma was more in spindle leaflets (8.9?×?10⁵ g of tissue) followed by roots (7.4?×?10⁵ g of tissue). Thus, a qPCR approach for detection and quantification of coconut phytoplasma was more advantageous than other PCR methods in terms of sensitivity and also reduced risk of cross contamination in the samples. Early diagnosis and quantification will pave way for the healthy coconut saplings selection and management under field conditions.

     

松材线虫侵染的马尾松人工林细根形态及生物量分异特征

中龄林的马尾松受松材线虫侵染后,林木生长、生理生化指标、群落多样性等会发生异质性变化,但是,针对患病林木地下细根的响应尚不清楚。本研究以松材线虫疫区患病马尾松和健康马尾松为研究对象,采用土柱法,分0-15 cm和15-30 cm土层,对细根进行分级研究,定量分析1-5级细根的形态、生物量以及养分元素,探讨松材线虫侵染的马尾松人工林细根形态、生物量以及养分元素的分异特征。结果表明:(1)患病马尾松人工林细根的健康状态与根长密度、生物量呈极显著正相关(P<0.01),低级根(如1级根)患病后,响应会更加强烈。(2)马尾松人工林患病后,细根有效磷、速效钾浓度会显著降低(P<0.05),而全氮、钙浓度会显著升高(P<0.05)。(3)松材线虫病使林分的土壤有机质含量显著高于健康林分(P<0.05),而土壤速效钾含量会显著低于健康林分(P<0.05)。以上结果表明,松材线虫侵染的马尾松人工林会在细根形态、细根养分和土壤养分上会发生特异性响应,揭示了松材线虫病对马尾松人工林地下细根的影响,旨在为松材线虫病防治提供一定参考。     

丛枝菌根真菌对黄瓜枯萎病的影响

Pot experiment was conducted to explore whether nursery inoculation of cucumber with Glomus etunicatum could alleviate fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum. Four-week-old seedlings inoculated with Glomus etunicatum were infected with F. oxysporum f. sp. cucumerinum by pouring conidial suspension. Biomass, contents of malonaldehyde (MDA), soluble sugar and free proline in roots, as well as the quantity of bacteria and fungi in rhizosphere were determined. The results indicated that the root dry weight of seedlings preinoculated with Glomus etunicatum increased by 9.3%; contents of soluble sugar and free proline in roots increased, and the quantity of fungi in rhizosphere decreased significantly. The disease incidence and disease index of Fusarium wilt were reduced. On the contrary, root dry weight of seedlings without inoculation with Glomus etunicatum was reduced by 28.0%. It is concluded that Glomus etunicatum is beneficial to biocontrol of Fusarium wilt of cucumber seedlings.     

丛枝菌根真菌对黄瓜枯萎病的影响

盆栽条件下播种黄瓜Cucumis sativus同时接种丛枝菌根真菌Glomus etunicatum,4周后对接种处理和对照黄瓜苗分别浇灌Fusairum oxysporum f.sp.cucumerinum分生孢子悬液,2周后测定幼苗生物量、根内丙二醛、可溶性糖与游离脯氨酸含量及根围真菌和细菌数量。结果表明:接种Glomus etunicatum根系干重增加了9.3%,提高了根内可溶性糖和游离脯氨酸含量,显著减少了根围真菌数量,降低了黄瓜枯萎病的发病率和病情指数。而不接种Glomus etunicatum的黄瓜苗根系干重减少了28.0%。研究认为AM真菌Glomus etunicatum对黄瓜枯萎病具有一定的生防价值。     

Proteomic analysis of the sea-island cotton roots infected by wilt pathogen Verticillium dahliae

Verticillium wilt of cotton is a vascular disease mainly caused by the soil-born filamentous fungus Verticillium dahliae. To study the mechanisms associated with defense responses in wilt-resistant sea-island cotton (Gossypium barbadense) upon V. dahliae infection, a comparative proteomic analysis between infected and mock-inoculated roots of G. barbadense var. Hai 7124 (a cultivar showing resistance against V. dahliae) was performed by 2-DE combined with local EST database-assisted PMF and MS/MS analysis. A total of 51 upregulated and 17 downregulated proteins were identified, and these proteins are mainly involved in defense and stress responses, primary and secondary metabolisms, lipid transport, and cytoskeleton organization. Three novel clues regarding wilt resistance of G. barbadense are gained from this study. First, ethylene signaling was significantly activated in the cotton roots attacked by V. dahliae as shown by the elevated expression of ethylene biosynthesis and signaling components. Second, the Bet v 1 family proteins may play an important role in the defense reaction against Verticillium wilt. Third, wilt resistance may implicate the redirection of carbohydrate flux from glycolysis to pentose phosphate pathway (PPP). To our knowledge, this study is the first root proteomic analysis on cotton wilt resistance and provides important insights for establishing strategies to control this disease.     

Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma

The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.     

Colonization of Fusarium Wilt‐Resistant and Susceptible Watermelon Roots by a Green‐Fluorescent‐Protein‐tagged Isolate of Fusarium oxysporum f.sp. niveum

Interactions between watermelon and a green fluorescent protein (GFP)‐tagged isolate of Fusarium oxysporum f.sp. niveum race 1 (Fon‐1) were studied to determine the differences in infection and colonization of watermelon roots in cultivars resistant to and susceptible to Fusarium wilt. The roots of watermelon seedlings were inoculated with a conidial suspension of the GFP‐tagged isolate, and confocal laser scanning microscopy was used to visualize colonization, infection and disease development. The initial infection stages were similar in both the resistant and susceptible cultivars, but the resistant cultivar responded differentially after the pathogen had penetrated the root. The pathogen penetrated and colonized resistant watermelon roots, but further fungal advance appeared to be halted, and the fungus did not enter the taproot, suggesting that resistance is initiated postpenetration. However, the tertiary and secondary lateral roots of resistant watermelon also were colonized, although not as extensively as susceptible roots, and the hyphae had penetrated into the central cylinder of lateral roots forming a dense hyphal mat, which was followed by a subsequent collapse of the lateral roots. The initial infection zone for both the wilt‐susceptible and wilt‐resistant watermelon roots appeared to be the epidermal cells within the root hair zone, which the fungus penetrated directly after forming appressoria. Areas where secondary roots emerged and wounded root tissue also were penetrated preferentially.     

The influence of normal tillage and of non-cultivation on Verticillium wilt of the hop

The influence of a non-cultivation (herbicide) regime on wilt incidence was compared with that of normal tillage in three observation trials on commercial hop farms. Non-cultivation resulted, on average, in 28% wilt control when wilt incidence was high. When wilt incidence was low there was a tendency for more wilt to occur with non-cultivation. The reduction in wilt under non-cultivation was probably mainly due to the virtually complete eradication throughout the year of alternative weed host plants, but other possible influential factors were: (i) the absence of root severance which could result in increased inoculum potential, (ii) changes in the pattern of hop root distribution, and (iii) the inhibiting action of simazine on root growth in surface soil resulting in reduced contact between roots and surface-borne inoculum. The lack of disease control with non-cultivation when wilt incidence was low was attributed to differential effects of the treatments on nitrogen metabolism or availability. The factors contributing to disease control under non-cultivation were considered to represent aspects of the process of sanitation, and the data were interpreted in terms of the sanitation ratio (sensu van der Plank). When wilt incidence was high the sanitation ratios at the three sites were similar. Modified cultural practices provide an important supplement to genetic resistance in the control of hop wilt. These practices (restricted use of nitrogen fertilizers and efficient sanitation with respect to the crop plant as well as to weeds) have proved economical and beneficial for hop culture in presence of wilt, under the threat of wilt, and in the absence of wilt.     

Interaction between Fusarium oxysporum F. vasinfectum and Cephalosporium maydis on cotton and maize

The interaction between Fusarium oxysporum (cause of cotton wilt) and Cephalosporium maydis (cause of maize late-wilt) on cotton roots is associated with an appreciable decrease in the severity of the cotton wilt disease. Reduction in infection is more pronounced when the latter fungus precedes the former in the soil than when they are inoculated simultaneously. C. maydis exerts little or no such effect when it follows F. oxysporum in the soil. C. maydis grows on the surface of cotton roots near growing points as a root-surface inhabitant. Dark red lesions are produced but these disappear, as does the fungus, when the root becomes hardened either naturally or in response to the growth of the fungus on the surface. The presence of the fungus is associated with increased production of root laterals. Cotton plants, including those which may appear healthy, show only mild internal symptoms of Fusarium infection when grown in soil inoculated with the two fungi, suggesting that the decreased severity of wilt is largely due to increased tolerance of the plants to infection with the disease as a result of increased number of root laterals. It is also possible that cotton roots with C. maydis on their surface become less suitable for the progress of F. oxysporum. F. oxysporum produces in culture a metabolite inhibitory to C. maydis. This may partly account for the little effect that the latter fungus exerts on the severity of wilt when it follows F. oxysporum in the soil. It appears that the interaction between F. oxysporum and C. maydis does not affect the pathogenicity of the latter fungus to maize.     

Induction of systemic resistance in chickpea against Fusarium wilt by Bacillus strains

Plant growth promoting rhizobacteria (PGPR) strains Rb29 (B. amyloliquefaciens MF352007), Bs1 (B. subtilis MF352017) and Bt1 (B. tequilensis MF352019) were tested for growth promotion and for their ability to induce systemic resistance against Fusarium wilt, a vascular disease of chickpea, using two methods that include whole plant and a split-root system. Bacillus strains and Fusarium oxysporum f. sp. ciceris (FOC) were inoculated on separate halves of roots of chickpea seedlings at the same time and then planted in separate pots either in superposition or one side of the other. All Bacillus strains systemically induced resistance against FOC, and significantly (p < 0.05) reduced the wilt disease by 98–100%. Application of Bacillus strains effectively enhanced plant growth, leading to increased plant height, root length, a fresh and dry weight of shoots and roots. These results help to explain the role of strains of Bacillus in growth promotion and biological control of Fusarium wilt in chickpea. This is the first report of systemic-induced resistance against Fusarium wilt in chickpea obtained by application of Bacillus strains to a root system spatially separated from the FOC-inoculated root.     

Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.     

A simple hydroponic culture method for the development of a highly viable root system in Arabidopsis thaliana

In the studies of nutritional absorption and metal toxicity in the root, it is important to grow plants without technical damage. We established a simple hydroponic culture system for Arabidopsis thaliana to obtain a healthy plant having a well-developed root system with many lateral roots. The phytotoxic effects of Cr, Cu, and Al ions were examined by FDA-PI staining using this culture system. The pattern of root inhibition varied with the ion, suggesting the usefulness of this culture system.     

A Simple Hydroponic Culture Method for the Development of a Highly Viable Root System in Arabidopsis thaliana

In the studies of nutritional absorption and metal toxicity in the root, it is important to grow plants without technical damage. We established a simple hydroponic culture system for Arabidopsis thaliana to obtain a healthy plant having a well-developed root system with many lateral roots. The phytotoxic effects of Cr, Cu, and Al ions were examined by FDA-PI staining using this culture system. The pattern of root inhibition varied with the ion, suggesting the usefulness of this culture system.     

香蕉植株根区土壤的真菌多样性分析

尖孢镰孢菌古巴专化型(Fusarium oxysporum f.sp.cubense)是香蕉枯萎病的病原菌,该菌是一种土壤习居菌,了解香蕉根区土壤中真菌多样性及镰孢菌属(Fusarium)真菌所占比例,对如何减少土壤中的病原菌、预防香蕉枯萎病的发生有重要的指导意义。该文通过采集不同宿根年限的香蕉健康植株和枯萎病植株的根区土壤,利用高通量测序技术测定土壤样品中的真菌种群。结果表明:(1)同一宿根年限的香蕉植株中,健康植株根区土壤中所获的reads及OTUs数量均高于枯萎病植株,说明健康植株根区土壤的真菌多样性丰富于枯萎病植株。(2)除了一年生香蕉枯萎病植株以担子菌门(Basidiomycota)为主外,其他土壤样品中均以子囊菌门(Ascomycota)为主,其中的丛赤壳科最高相对丰度来自三年生健康植株的根区土壤(26.02%),其次是五年生的枯萎病植株根区土壤(15.56%)。(3)在丛赤壳科中,镰孢菌属在三年生健康植株土壤中的相对丰度最高(2.54%),在其他样品中的相对丰度在0.1%～0.65%之间;在镰孢菌属中,腐皮镰孢菌(Fusarium solani)的相对丰度(0～1.59%...     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.     

Histological changes in guava root during wilting induced by Fusarium spp.

Wilt is the most destructive disease of guava in India. Fusarium oxysporum f. sp. psidii and Fusarium solani are reported as most commonly isolated pathogens and are considered to be associated singly or in combination with roots of wilt affected plants of guava (Psidium guajava L.). Histopathological observations were made of the roots of wilt affected guava plants in the present investigation. The observations of wilted guava root showed disintegration/necrosis of the epidermal tissue, cortex tissue and vascular bundle cells. In T.S. of root of wilted plant the normal shape of the epidermis was disrupted and there was breaking and opening in the epidermis through which pathogen may enter in the host tissue. Necrosis of the internal tissue and vascular bundle restricts the movement of water and nutrient and thus results wilting.     

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.     

Studies on thermoprotection induced by heavy metal ions in spring barley seedlings

Barley seedlings (Hordeum vulgare L., cv. Fatran) were pretreated with various concentrations of five heavy metal ions (Zn, Cu, Fe, Mg, Mn) for 3 d. When the subsequent heat shock was administered for 2 h, the heavy metal ions had thermoprotective effect against the sub-lethal (40 °C) and lethal (45 °C) temperature stresses, which were otherwise lethal to control (water grown) seedlings. The effectiveness of each of the heavy metal ions was different, the most effective being Cu. The level of protection provided by these heavy metal ions was dependent on both the time and the concentration that plants were exposed to them. The greatest differences were recorded in the thermotolerance mediated by applied metal ions in the shoot and root cells. Thermotolerance was exhibited by both the shoot and root of pretreated seedlings, even though the heavy metal stresses were applied solely to the roots.