Arabidopsis thaliana,an experimental host for tomato yellow leaf curl disease‐associated begomoviruses by agroinoculation and whitefly transmission

Tomato yellow leaf curl disease is one of the most devastating viral diseases affecting tomato crops worldwide. This disease is caused by several begomoviruses (genus Begomovirus, family Geminiviridae), such as Tomato yellow leaf curl virus (TYLCV), that are transmitted in nature by the whitefly vector Bemisia tabaci. An efficient control of this vector‐transmitted disease requires a thorough knowledge of the plant–virus–vector triple interaction. The possibility of using Arabidopsis thaliana as an experimental host would provide the opportunity to use a wide variety of genetic resources and tools to understand interactions that are not feasible in agronomically important hosts. In this study, it is demonstrated that isolates of two strains (Israel, IL and Mild, Mld) of TYLCV can replicate and systemically infect A. thaliana ecotype Columbia plants either by Agrobacterium tumefaciens‐mediated inoculation or through the natural vector Bemisia tabaci. The virus can also be acquired from A. thaliana‐infected plants by B. tabaci and transmitted to either A. thaliana or tomato plants. Therefore, A. thaliana is a suitable host for TYLCV–insect vector–plant host interaction studies. Interestingly, an isolate of the Spain (ES) strain of a related begomovirus, Tomato yellow leaf curl Sardinia virus (TYLCSV‐ES), is unable to infect this ecotype of A. thaliana efficiently. Using infectious chimeric viral clones between TYLCV‐Mld and TYLCSV‐ES, candidate viral factors involved in an efficient infection of A. thaliana were identified.     

Phenotypic expression, stability, and inheritance of a recessive resistance to monopartite begomoviruses associated with tomato yellow leaf curl disease in tomato

Tomato-infecting begomoviruses comprise a complex of monopartite and bipartite virus species that cause severe yield and quality losses worldwide. Therefore, the availability of wide spectrum resistance for begomovirus control is desirable. However, limited sources of resistance are available. In this study, three tomato inbred lines with resistance to bipartite begomoviruses of Brazil were tested for resistance to monopartite begomoviruses associated with the tomato yellow leaf curl disease (TYLCD). Stable resistance to Tomato yellow leaf curl virus was observed either by inoculation with Bemisia tabaci or with Agrobacterium tumefaciens using an infectious clone. The resistance resulted in a complete absence of TYLCD symptoms and restricted virus accumulation. Further studies performed with the line '468-1-1-12' indicated that the resistance was also effective against three other virus species associated with TYLCD, indicating wide spectrum resistance of this source. Quantitative genetics analyses suggested that a major recessive locus with epistatic interactions is controlling the resistance to TYLCD in '468-1-1-12', which could facilitate introgression of this trait into elite tomato lines. The resistance was stable under field conditions with high TYLCD pressure. Mild symptoms could be observed in these conditions, and recovery from disease and from virus infection suggested an active host antiviral defense mechanism. The differential reaction of '468-1-1-12' against a number of TYLCD-associated viruses and artificial chimeras between them allowed to identify a region of the virus genome that presumably contains a virus determinant for breaking the resistance to infection observed in '468-1-1-12'.     

A new approach to evaluate relative resistance and tolerance of tomato cultivars to begomoviruses causing the tomato yellow leaf curl disease in Spain

A simple procedure to evaluate relative resistance and tolerance of tomato cultivars to the begomoviruses causing tomato yellow leaf curl (TYLC) disease in Spain was developed. To estimate the resistance and tolerance levels of a cultivar, several formulae were developed based on the ratio of infected plants, virus titre (estimated by tissue–print hybridization) and symptom intensity. The formulae were applied to five commercial tomato cultivars (Amoretto, Birloque, Royesta, Tovigreen and Ulises) naturally infected by TYLC viruses. The analyses showed that Ulises, Birloque and Tovigreen exhibited a moderate resistance, and Ulises was also highly tolerant. There was a positive correlation between symptom intensity and virus titre in infected plants, suggesting that the hybridization technique could also be used as an early estimator of tolerance. Finally, molecular hybridization and nucleotide sequence analyses of the begomovirus intergenic region showed that the local TYLC virus population consisted of a single species, Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Israel), with low genetic variation (nucleotide identity between isolates higher than 97%).     

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.     

Genetic diversity and mixed infections of begomoviruses infecting tomato, pepper and cucurbit crops in Nicaragua

Begomoviruses were detected in Nicaraguan fields of tomato ( Lycopersicon esculentum ) and adjacently growing plants of pepper ( Capsicum annuum ), chilli pepper ( C . baccatum ), cushaw ( Cucurbita argyrosperma ) and Mexican fireplant ( Euphorbia heterophylla ) using polymerase chain reaction (PCR) and universal begomovirus primers. All tomato and Mexican fireplant plants showing symptoms were infected with begomoviruses, while only 30–46% of the pepper, chilli pepper and cushaw plants showing symptoms tested virus-positive. No begomoviruses were found in potato. The virus species were provisionally identified by sequencing 533 bp of the viral coat protein gene ( AV1 ). Tomato severe leaf curl virus (ToSLCV), Tomato leaf curl Sinaloa virus (ToLCSinV) and Pepper golden mosaic virus (PepGMV) were found to infect both tomato and pepper. A new provisional species designated Tomato leaf curl Las Playitas virus (ToLCLPV) was detected in a tomato plant. Squash yellow mottle virus (SYMoV) and PepGMV were found in cucurbits, the latter for the first time in this host. Euphorbia mosaic virus (EuMV) was detected in Mexican fireplant. Sequencing of a larger number of PCR-amplified clones from selected plants revealed intraspecific viral sequence variability, and also multiple begomovirus infections which could represent up to three species in a single tomato or cushaw plant. Phylogenetic grouping of virus sequences did not correlate with the host of origin.     

Biochemical and molecular mechanisms of diafenthiuron resistance in the whitefly,Bemisia tabaci

B-biotype Bemisia tabaci has developed high levels of resistance to many insecticides. To investigate the risks and explore possible mechanisms of resistance to diafenthiuron in B. tabaci, a 32.8-fold diafenthiuron-resistant strain (R-DfWf) was established after selection for 36 generations compared with the susceptible strain (S-Lab). Biochemical assays showed that the activity of cytochrome P450 towards p-NA was significantly higher (4.37-fold higher) in the R-DfWf strain than in the S-Lab strain. Similarly, the carboxylesterase (COE) activity and glutathione S-transferase (GST) activity were also significantly higher (3.12- and 1.83-fold higher, respectively) in the R-DfWf strain than in the S-Lab strain. The expression of five of seven P450 genes was significantly higher (>3-fold) in the R-DfWf strain than in the S-Lab strain. The expression of COE2 was significantly higher (>2.5-fold) in the R-DfWf than in the S-Lab strain. The expression of GST and GST2 was significantly higher (>2.3-fold) in the R-DfWf than in the S-Lab. Thus, cytochrome P450, COE and GST may appear to be responsible for the resistance to diafenthiuron in B. tabaci. It is also valuable for usage of insecticides for resistance management and control of this species.     

Cytological aspects of compost-mediated induced resistance against fusarium crown and root rot in tomato

ABSTRACT The potential of a pulp and paper mill residues compost for the control of crown and root rot of greenhouse-grown tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici was ultrastructurally investigated. Peat moss amended with compost substantially reduced disease-associated symptoms. Addition of Pythium oligandrum to either peat moss alone or peat moss amended with compost resulted in a considerable reduction in disease incidence compared with controls grown in peat moss alone. Histological and cytological observations of root samples from Fusarium-inoculated plants revealed that the beneficial effect of compost in reducing disease symptoms is associated with increased plant resistance to fungal colonization. One of the most prominent facets of compost-mediated induced resistance concerned the formation of physical barriers at sites of attempted fungal penetration. These structures, likely laid down to prevent pathogen ingress toward the vascular elements, included callose-enriched wall appositions and osmiophilic deposits around the sites of potential pathogen ingress. Invading hyphae, coated by the osmiophilic material, showed marked cellular disorganization. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin was altered in severely damaged hyphae. A substantial increase in the extent and magnitude of the cellular changes induced by compost was observed when P. oligandrum was supplied to the potting substrate. This finding corroborates the current concept that amendment of composts with specific antagonists may be a valuable option for amplifying their beneficial properties in terms of plant disease suppression.     

Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato

A reproducible and accurate procedure, based on HPLC analysis, has been developed to determine simultaneously acibenzolar‐S‐methyl (CGA 245 704) and its acid derivative (CGA 210 007) in tomato leaves. The limit of detection and quantification of the method are 0.015 and 0.15 mg litre^?1 for CGA 245 704 and 0.030 and 0.30 mg litre^?1 for CGA 210 007. In tomato plants treated with 250 µM CGA 245 704, it was found that the inducer rapidly translocates from treated leaves (cotyledons, 1st and 2nd) to untreated leaves (3rd to 5th), with the maximum translocation (40% of the total quantity found) occurring 8 h after the treatment. CGA 245 704 residues decreased as time elapsed in both treated and untreated tomato leaves, reaching negligible values 72 h after treatment. The acid derivative, CGA 210 007, was formed in tomato plants as early as 2 h after CGA 245 704 treatment, albeit only in the treated leaves. CGA 210 007 residues decreased in treated tomato leaves with a trend similar to that observed for CGA 245 704. Treatment of tomato plants with CGA 245 704 or CGA 210 007 at 250 µM systemically protected the plants against Pseudomonas syringae pv tomato attacks, the causal agent of bacterial speak disease. Evidence of this were reductions in the degree of infection, the bacterial lesion diameter and the bacterial growth in planta. Since neither CGA 245 704 nor CGA 210 007 inhibited bacterial growth in vitro and the protection against bacterial speak of tomato was observed when the two compounds were completely degraded, the protection must be due to the activation of the plant's defence mechanisms. © 2001 Society of Chemical Industry     

云南怒江番茄种植新区番茄病毒检测及病毒种传特性

病毒病对番茄生产造成严重危害, 近年来在番茄种植新区发生严重, 疑似为种子带毒传播。本研究通过对云南省怒江州番茄种植新区的番茄病毒病样品采用RNA-seq高通量测序, RT-PCR验证的方法检测病毒种类;对番茄病果种子进行超薄切片制样透射电子显微镜观察, 将病果种子播种后对种苗进行RT-PCR带毒检测。结果表明, RNA-seq高通量测序及RT-PCR检测到的病毒有番茄环纹斑点病毒(tomato zonate spot orthotospovirus, TZSV)、番茄黄斑驳相关病毒(tomato yellow mottle-associated virus, TYMaV)、辣椒脉斑驳病毒(chili veinal mottle virus, ChiVMV)、南方番茄病毒(southern tomato virus, STV)。透射电镜观察到种胚细胞及胚乳细胞中分布典型的正番茄斑萎病毒属Orthotospovirus病毒粒体。病果种子播种28 d后的种苗具有病毒病症状, 通过RT-PCR检出TZSV、ChiVMV、STV, 检出率分别为60%、100%、80%。上述研究结果为TZSV通过种子传播提供了有利的证据, 并为源头防控番茄病毒病提供了依据。     

中国番木瓜曲叶病毒在南宁的发生及其烟粉虱传播和中间寄主初步研究

番木瓜曲叶病毒病在广西南宁市郊区发生严重并造成很大经济损失,经2002-2004年调查,重病果园发病率高达47.23%-100%,染病番木瓜矮缩,叶片向下卷曲,叶背部叶脉增生,叶柄扭曲,早期染病植株无法开花结果。番木瓜曲叶病毒病可以通过烟粉虱传染,但是,室内人工传染效率很低,仅为8.33%;用PCR方法从表现黄脉症状的胜红蓟(Ageratum conyzoides)和田麻(Corchoropsis tomentosa)及表现黄曲叶症状的番茄(Lycopersicon escu-lentum)中检出中国番木瓜曲叶病毒(PaLCuCNV),证明这3种植物是番木瓜曲叶病毒病的中间寄主。     

Tracing the geminivirus-whitefly transmission pathway by polymerase chain reaction in whitefly extracts, saliva, hemolymph, and honeydew

ABSTRACT A membrane feeding system and polymerase chain reaction (PCR) were used to track squash leaf curl virus (SLCV) DNA in whole whitefly body extracts and in saliva, honeydew, and hemolymph of its whitefly vector, Bemisia tabaci, and a whitefly nonvector, Trialeurodes vaporariorum. SLCV ingestion was monitored by PCR in whiteflies that were given acquisition access periods (AAPs) ranging from 0.5 to 96 h on virus-infected plants. SLCV detection by PCR in whole body extracts was considered reflective of virus ingestion. As whiteflies were given longer AAPs, the number of whiteflies that ingested SLCV increased. SLCV DNA was detected in honeydew of vector and nonvector whiteflies, indicating that virions, viral DNA, or both passed unimpeded through the digestive system. SLCV DNA was detected in saliva and hemolymph of B. tabaci, but not in these fractions from nonvector whiteflies, despite virus ingestion by both. Although vector and nonvector whiteflies both ingested SLCV, only in the vector, B. tabaci, did virus cross the gut barrier, enter the hemolymph, or pass into the salivary system. These results suggest that digestive epithelia of nonvector whiteflies did not permit SLCV passage from the gut to hemocoel, whereas virus effectively crossed the analogous gut barrier in vector whiteflies.     

Evaluation of two methods for the protection of tomato crops against the tomato leafminer Tuta absoluta (Meyrick) under greenhouses in Tunisia

The evaluation of two methods of plant protection in tomato crops cultivated in greenhouses against Tuta absoluta (Meyrick) in the region of Teboulba in Tunisia was performed. This study evaluated the use of insect‐proof screens alone or in combination with one sex pheromone water trap compared to a control greenhouse not equipped with either. The study confirms that the use of one sex pheromone water trap combined with insect‐proof covering of doors and aeration openings is sufficient to guarantee good crop protection. This combined control system allowed a low density of the pest (less than 2 individuals per leaf) to be maintained and a mean number of mines below 1 mine per leaf.     

High incidence of seed transmission of Papaya ringspot virus and Watermelon mosaic virus, two viruses newly identified in Robinia pseudoacacia

Black locust (Robinia pseudoacacia L.) is an ornamental legume tree susceptible to several viruses. Virus diseases of black locust often go unrecognized making infected trees a prime inoculum source, not only for legumes, but also other plant species. In this communication we report, for the first time, two viruses, Papaya ringspot virus and Watermelon mosaic virus, associated with disease in black locust. Isolates of both viruses were partially characterized and seed transmission was examined. The high percentage of seed transmission suggests that this may be an important avenue for virus dissemination.     

First screening for resistance in Argentinian cultivars against Pyrenophora teres F. maculata,a recently reported pathogen in wheat

European Journal of Plant Pathology - The Pyrenophora genus involves an extremely important group of fungal plant pathogens, causing some of the most devastating disease epidemics of cereal plants...     

Phytoplasma transmission by in vitro graft inoculation as a basis for a preliminary screening method for resistance in fruit trees

In vitro grafting was tested as a technique for inoculating Prunus rootstock Prunus marianna GF 8-1 with European stone fruit yellows (ESFY) phytoplasmas and apple rootstock Malus pumila MM106 with apple proliferation (AP) phytoplasmas. In vitro shoot cultures of ESFY-infected Prunus marianna GF 8-1 and AP-infected Malus pumila MM106 were used as graft inoculum to transmit the phytoplasmas to the respective healthy rootstock. Phytoplasma transmission was assessed after a graft contact of 1, 2 or 3 months. Healthy autografts were used as controls to monitor parameters of in vitro grafting. Successful graft union formation ranged from 58 to 79% irrespective of the plant species and the sanitary state of the graft. Pathogen-specific polymerase chain reaction (PCR) was used to test the inoculated rootstocks for the presence of ESFY and AP phytoplasmas, respectively. The rate of ESFY phytoplasma transmission in successful Prunus -grafts increased from 69 to 94% with the time of contact. AP phytoplasma transmission to Malus occurred in 80 to 97% of successful grafts. To our knowledge this is the first report of phytoplasma transmission by grafting in vitro . The results provide a good basis for the establishment of a preliminary in vitro screening method for phytoplasma resistance in Prunus and Malus .     

Evidence for two predominant viral lineages,recombination and subpopulation structure in begomoviruses associated with yellow vein mosaic disease of okra in India

Yellow vein mosaic disease (YVMD) caused by whitefly‐transmitted begomoviruses is an economically significant viral disease of okra. In this study, a survey of begomoviruses associated with YVMD was carried out in eight states and two union territories of India. A total of 92 full‐length DNA‐A components were sequenced and characterized. Sequence comparisons and population structure analysis revealed the existence of four begomovirus species. Two novel species were detected with several recombinationally derived genome fragments that probably originated from begomoviruses known to infect malvaceous and non‐malvaceous hosts. Among the four species, Bhendi yellow vein Maharastra virus (BYVMaV) and Bhendi yellow vein Madurai virus (BYVMV) were found to be predominant in okra, with BYVMV having a pan‐India distribution. There was evidence for a high degree of genetic variability and subpopulation structure within these four species. Neutrality tests suggested the occurrence of purifying selection acting upon these populations. The results of the current study have uncovered the diversity and genetic structure of okra‐infecting begomoviruses in India and generated potentially useful information for developing management strategies for YVMD.     

北京地区蔬菜烟粉虱种群动态及其对烟碱类杀虫剂的抗药性监测

为了明确北京地区蔬菜烟粉虱的种群发展动态及对常用烟碱类杀虫剂的抗药性,为烟粉虱防治适期的确定及高效杀虫剂的选择提供基础资料及科学指导,2009年调查了北京地区春茬和秋茬蔬菜烟粉虱的种群动态,并监测了该地区2009-2011年度烟粉虱种群对3种烟碱类杀虫剂的抗药性。结果表明,春茬蔬菜烟粉虱在5月中旬种群快速上升,持续到春茬拉秧;秋茬蔬菜烟粉虱在塑料棚内9月上旬种群数量即开始上升,露地出现时间较晚,10月中旬后种群数量下降;塑料棚内种群数量明显高于露地蔬菜烟粉虱数量。烟粉虱2009年对吡虫啉和噻虫嗪的抗药性很高,2010年和2011年抗性程度显著下降,LC50处于133.94～251.16mg/L,属中等抗性水平;而对新型杀虫剂烯啶虫胺的抗药性在近3年内持续升高,抗性倍数由6.68倍升至83.62倍,即由低抗性水平升至高抗性水平。上述调查结果表明,北京地区蔬菜烟粉虱的防治应掌握春季5月中旬前、秋季9月中旬前种群处于低密度时进行。烟粉虱对新型杀虫剂烯啶虫胺的抗药性在近三年内快速上升,生产中应注意药剂的轮换使用。     

Studies of canker and dieback of oak tree in China,with two Cytospora species described

Oak (Quercus sp.) is an excellent tree species as a windbreak, for water conservation, and for fireproofing in forests in China. However, several trees of this genus were found to be suffering from various fungal diseases. In this study, we evaluated 15 fungal pathogens that can cause dieback and canker disease in oak in China, and discovered two Cytospora species. They were identified as Cytospora quercinum sp. nov. and C. vinacea, based on detailed morphological comparisons and phylogenetic analysis of ITS, LSU, act, rpb2, tef1-α, and tub2 loci. This study is the first record of C. quercinum and C. vinacea as causal agents of dieback in oak based on pathogenicity tests conducted on 2-year-old plants in a greenhouse. In addition, this study also revealed the influence of different conditions on the growth rate of mycelia. Mycelial growth of C. quercinum and C. vinacea occurred at optimum temperatures of 20.1 and 20.8 °C, and optimum pH of 5.4 and 5.3, respectively. For these two species, utilization of glucose and fructose was highly efficient, and sucrose was the least efficient. The habitat of Quercus mongolica indicated that more attention and management are needed to prevent the occurrence of Cytospora disease in the summer in north-eastern China, and in spring and autumn in eastern and northern China. This study contributes to the understanding of the species causing canker or dieback diseases in important economic forest trees, and provides useful information for effective disease management of oak trees in China.