Discrimination between Virulent and Attenuated Isolates of Tomato mosaic virus by Restriction Fragment Length Polymorphism

The three missense mutations on the gene for the 130-K protein of Tomato mosaic virus (ToMV) L₁₁A have been thought to be responsible for the attenuation of its virulence. The Eco47I RFLP detecting the missense mutation at 2349 successfully discriminated L₁₁A and its derivative attenuated isolates from ToMV virulent ones. RFLP analysis and mismatch amplification assay detecting the missense mutations at 1117 and 2754, respectively, could not discriminate some of the attenuated isolates from the virulent ones. These results indicated that, of the three missense mutations, only the one at 2349 was conserved in all the L₁₁A-derivative attenuated isolates. Received 16 March 2001/ Accepted in revised form 22 June 2001     

The coat protein of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> L<Subscript>11</Subscript>Y is associated with virus-induced chlorosis on infected tobacco plants

The L₁₁Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L₁₁Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L₁₁Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L₁₁Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L₁₁Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L₁₁Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L₁₁Y. Fewer virus particles accumulated in L₁₁Y-infected protoplasts, and many of them were shorter-than-full-length. The nucleotide sequence data reported is available in DDBJ/EMBL/GenBank databases as accession AB355139.     

Viruses of pepper in plastic houses in Crete

During a survey of virus diseases affecting pepper grown in plastic houses in Crete, during 1984–1986, tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) were detected. The most common virus was TMV, being present in samples of alle pepper cultivars carrying the L₁ resistance gene, while ToMV was isolated only from susceptible pepper cultivars. According to responses ofCapsicum spp. the isolates from 640 samples checked were classified into three pathotypes: P₀, P_1.2 and P_1.2.3. Results of this study show that P_1.2 represents at present the major threat to the Cretan pepper industry.Samenvatting Bij een in de jaren 1984–1986 gehouden inventarisatie van virusziekten in paprika in plastic-foliekassen op Kreta werd zowel het tabaksmozaïekvirus (TMV) als het tomatemozaïekvirus (ToMV) aangetoond. TMV kwam zeer algemeen voor: het werd aangetroffen in alle monsters van de paprikarassen met het resistentiegen L₁. ToMV werd alleen geïsoleerd uit planten van vatbare paprikarassen. Volgens de waargenomen symptomen die de isolaten van 640 monsters opCapsicum spp. vertoonden, konden de isolaten in drie pathotypen, nl. P₁, P_1,2 en P_1,2,3, worden geklassificeerd. Uit het onderzoek is gebleken dat P_1,2 de belangrijkste bedreiging vormt voor de teelt van paprika op Kreta.     

新疆番茄病毒病检测及南方番茄病毒全基因组序列测定与传播特性分析

为了解新疆番茄上病毒病的发生情况,利用一步法RT-PCR技术检测了南北疆番茄上南方番茄病毒(Southern tomato virus,STV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)以及马铃薯Y病毒(Potato virus Y,PVY)的感染情况,并利用分段克隆的方法进行全基因组测序,通过RT-PCR方法检测健康植株与携带病毒植株杂交育种的F1代植株带毒率以分析STV的种子传播特性。结果显示,新疆番茄上CMV、STV、ToMV和PVY在北疆的检出率分别为52%、37%、27%和14%;在南疆的检出率分别为79%、60%、69%和0;且以STV、CMV及ToMV的复合侵染为主。从我国加工番茄上首次获得了长3 437 nt的STV SHZ-1核苷酸序列,序列比对分析发现其与已报道STV只有1~9个核苷酸的变异,且序列变异与地域无相关性。分析杂交F1代加工番茄植株上STV的传播特性,发现其除可由种子传播外,也可通过花粉传播。表明STV是侵染新疆番茄的主要病毒之一,且该病毒可通过种子或杂交育种途径进行传播。     

Effects of foliar sprayed calcium sources onTomato mosaic virus (ToMV) infection in tomato plants grown in greenhouses

Spray solutions containing 0.3% Ca which were prepared from four different calcium sources were foliar-sprayed on greenhouse-grown tomato plants, infected with theTomato mosaic virus Tobamovirus (ToMV) or not. ToMV-infected and uninfected control groups were sprayed with distilled water. Growth and macronutrient (N, P, K, Ca and Mg) composition of tomato plants as well as virus concentration and its relative infectivity were investigated in treated and untreated plants. The Ca sprays were applied three times: on the same day as inoculation, and 15 and 30 days after inoculation. Virus concentration in tomato plants generally decreased with foliar-sprayed Ca. Virus concentration (DAS-ELISA absorbance) was reduced by foliar-sprayed Ca, but plants remained infected. At the same time, tissue Ca concentrations increased significantly with foliar-applied Ca, with the exception of CaNO₃·4H₂O+0.05M Na-EDTA. ToMV reduced the fresh and dry weights and Ca concentrations of tomato plants, but significantly raised P concentration in the tissue. Neither virus inoculation nor foliar Ca applications affected N and Mg concentrations in tomato plants. The foliar-applied Ca from all the sources gave K concentrations similar to those of control plants. http://www.phytoparasitica.org posting Jan. 26, 2007.     

Indexing system for<Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) in commercial tomato seed lots

An indexing system for detectingTomato mosaic virus (ToMV) in commercial tomato seed lots is described. Factors associated with the procedure were analyzed and the following standard two-step working scheme is proposed: (i) mass screening by ELISA for the presence of the virus; (ii) evaluation of virus infectivity within the infested seed lots. A threshold of 10 ng ml⁻¹ was determined for detection of purified ToMV by either ELISA or plant inoculation.Nicotiana tabacum cv. Xanthi NN was found to be a highly sensitive local lesion assay plant for the detection of ToMV. A positive ELISA threshold (1.3 times above the non-specific background) was set for seed samples taken from commercial seed lots by testing the same samples by both ELISA and a bioassay. http://www.phytoparasitica.org posting July 14, 2004.     

Phylogeny of Egyptian isolates of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> (CMV) and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> (ToMV) infecting <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Four Cucumber mosaic virus (CMV) (CMV-HM 1–4) and nine Tomato mosaic virus (ToMV) (ToMV AH 1–9) isolates detected in tomato samples collected from different governorates in Egypt during 2014, were here characterized. According to the coat protein gene sequence and to the complete nucleotide sequence of total genomic RNA1, RNA2 and RNA3 of CMV-HM3 the new Egyptian isolates are related to members of the CMV subgroup IB. The nine ToMV Egyptian isolates were characterized by sequence analysis of the coat protein and the movement protein genes. All isolates were grouped within the same branch and showed high relatedness to all considered isolates (98–99%). Complete nucleotide sequence of total genomic RNA of ToMV AH4 isolate was obtained and its comparison showed a closer degree of relatedness to isolate 99–1 from the USA (99%). To our knowledge, this is the first report of CMV isolates from subgroup IB in Egypt and the first full length sequencing of an ToMV Egyptian isolate.     

The reproductive potential of the root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> is affected by selection for virulence against major resistance genes from tomato and pepper

The emergence of virulent root-knot nematode populations, able to overcome the resistance conferred by some of the resistance genes (R-genes) in Solanaceous crops, i.e., Mi(s) in tomato, Me(s) in pepper, may constitute a severe limitation to their use in the field. Research has been conducted to evaluate the durability of these R-genes, by comparing the reproduction of several laboratory-selected and wild virulent Meloidogyne incognita isolates, on both susceptible and resistant tomatoes and peppers. We first show that the Me1 R-gene in pepper behaves as a robust R-gene controlling avirulent and virulent Me3, Me7 or Mi-1 isolates. Although the reproductive potential of the virulent isolates was highly variable on susceptible and resistant plants, we also confirm that virulence is highly specific to a determined R-gene on which selection has occurred. Another significant experimental result is the observation that a reproductive fitness cost is associated with nematode virulence against Mi-1 in tomato and Me3 and Me7 in pepper. The adaptative significance of trade-offs between selected characters and fitness-related traits, suggests that, although the resistance can be broken, it may be preserved in some conditions if the virulent nematodes are counter-selected in susceptible plants. All these results have important consequences for the management of plant resistance in the field.     

Effectiveness and profitability of the <Emphasis Type="Italic">Mi</Emphasis>-resistant tomatoes to control root-knot nematodes

Experiments were conducted to determine the effectiveness and profitability of the Mi-resistance gene in tomato in suppressing populations of Meloidogyne javanica in a plastic-house with a natural infestation of the nematode. Experiments were also conducted to test for virulence and durability of the resistance. Monika (Mi-gene resistant) and Durinta (susceptible) tomato cultivars were cropped for three consecutive seasons in non-fumigated or in soil fumigated with methyl bromide at 75 g m^–2 and at a cost of 2.44 euros m^–2. Nematode densities were determined at the beginning and end of each crop. Yield was assessed in eight plants per plot weekly for 6 weeks. The P_f/P_i values were 0.28 and 21.6 after three crops of resistant or susceptible cultivars, respectively. Growth of resistant as opposed to susceptible tomato cultivars in non-fumigated soil increased profits by 30,000 euros ha^–1. The resistant Monika in non-fumigated soil yielded similarly (P > 0.05) to the susceptible Durinta in methyl bromide fumigated soil but the resistant tomato provided a benefit of 8800 euros ha^–1 over the susceptible one because of the cost of fumigation. Selection for virulence did not occur, although the nematode population subjected to the resistant cultivar for three consecutive seasons produced four times more eggs than the population on the susceptible one. Such a difference was also shown when the resistant cultivar was subjected to high continuous inoculum pressure for 14 weeks. The Mi-resistance gene can be an effective and economic alternative to methyl bromide in plastic-houses infested with root-knot nematodes, but should be used in an integrated management context to preserve its durability and prevent the selection of virulent populations due to variability in isolate reproduction and environmental conditions.     

Quantitative approach for the early detection of selection for virulence of Meloidogyne incognita on resistant tomato in plastic greenhouses

Resistant tomato cultivars are an important tool to control Meloidogyne spp., which cause the highest yield losses attributed to plant‐parasitic nematodes. However, the repeated cultivation of Mi resistant cultivars can select virulent populations. In the present study, the susceptible tomato cv. Durinta and the resistant cv. Monika were cultivated from March to July in a plastic greenhouse for 3 years to determine the maximum multiplication rate, maximum nematode density, equilibrium density, relative susceptibility and population growth rate of M. incognita; these were used as proxy indicators of virulence and yield losses. The values of population dynamics and growth rate on the resistant tomato increased year by year and were higher when it was repeatedly cultivated in the same plot compared to when it was alternated with the susceptible cultivar and the level of resistance decreased from very to moderately resistant. The relationship between the nematode density at transplanting (P_i) and the relative yield of tomato fitted to the Seinhorst damage model for susceptible, but not resistant, cultivars. The tolerance limit and the relative minimum yield were 2–4 J2 per 250 cm³ of soil and 0.44–0.48, respectively. The tomato yield did not differ between cultivars at low P_i, but it did at higher P_i values, at which the resistant yielded 50% more than the susceptible. This study demonstrates the utility of population dynamics parameters for the early detection of selection for virulence in Meloidogyne spp., and that three consecutive years were not sufficient to select for a completely virulent population.     

Characterization of Rehmannia mosaic virus isolated from chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) in Japan

In 2010, severe necrotic mosaic disease and fruit distortion were observed on greenhouse-grown chili pepper (Capsicum annuum cv. Fushimi-amanaga) plants in Kyoto Prefecture, Japan. Electron microscopic imaging and genomic RNA sequencing indicated that the virus responsible was a new isolate of Rehmannia mosaic virus (ReMV), which had not been previously reported in Japan. Although ReMV systemically infected many Solanaceae species, including chili pepper and tomato (Solanum lycopersicum), tobamovirus-resistance genes from species of Capsicum (L ^1a , L ² , L ³ , and L ⁴ ) and tomato (Tm-1, Tm-2, and Tm-2 ^a ) conferred resistance against ReMV.     

Interference with cell-to-cell movement of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> by transient overexpression of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> KELP homologs from different plant species

The KELP protein of Arabidopsis thaliana (AtKELP), which binds to the Tomato mosaic virus (ToMV) movement protein in vitro, can interfere with cell-to-cell movement of the virus under transient overexpression conditions. In this study, we constructed expression plasmids encoding a fluorescent protein fused to AtKELP or to its homolog from Brassica, Nicotiana, Solanum, or Oryza species. When expressed transiently, all the AtKELP homologs inhibited the cell-to-cell movement of ToMV with different efficiencies. The results of domain swapping between two selected AtKELP homologs suggest the importance of combination between the N- and C-terminal half regions for high inhibitory function.     

Effects of isolate virulence and host susceptibility on development of early blight (Alternaria solani) on tomato*

Early blight of tomato (Lycopersicon esculentum) caused by Alternaria solani has the potential to become one of the most serious diseases throughout the tomato‐producing regions of Greece. Controlled environment experiments were conducted to study the virulence of A. solani isolates and the susceptibility of commercial tomato cultivars and hybrids to early blight. The isolates used, derived from naturally infected tomato plants during the period 1997/1998, differed significantly (P>0.05) in the rate of mycelial growth as well as in their ability to sporulate in vitro. No correlation (R²= 0.33) was found between mycelial growth and conidia production. Isolates of A. solani were virulent to young tomato plants (cv. Ace 55VF), although they differed significantly (P>0.05) in the intensity of symptoms produced on leaves, stems, petioles and flowers. Defoliation was linearly related (R²= 0.87) to the percentage of leaf area with symptoms. Twenty‐three tomato cvs. or F₁ hybrids were evaluated for their susceptibility to early blight. The cultivars or hybrids were arbitrarily categorized as immune, highly tolerant, tolerant, moderately tolerant, susceptible and highly susceptible based on a percent disease index range: 0%, 1‐9%, 10‐24%, 25‐49%, 50‐74% and 75% or more respectively. None of the cultivars or hybrids tested was immune or tolerant to A. solani infection.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Occurrence of virulent root-knot nematode populations on tomatoes bearing the <Emphasis Type="Italic">Mi</Emphasis> gene in protected vegetable-growing areas of Turkey

Root-knot nematodes (Meloidogyne spp.; RKN) are one of the most important pathogens of vegetables in Turkey. Assessing the existing virulent RKN populations is of importance for pathogen mapping in the west Mediterranean region of Turkey. Therefore, 95 populations of RKN were collected from different protected vegetable-growing locations in the region. Pure cultures were obtained and identified by means of species-specific primers. Virulence of the populations against the Mi-1 gene conferring resistance to Meloidogyne incognita, M. javanica and M. arenaria was determined according to their egg masses and gall rating on resistant and susceptible tomato varieties. Results showed that seven populations of M. incognita and six populations of M. javanica were able to overcome the resistance controlled by the Mi-1 gene. The frequency of virulent populations of M. incognita and M. javanica collected from different protected-grown vegetables was 11.7% and 21.4%, respectively. To our knowledge, this is the first report of populations of RKN virulent to the Mi-1 gene in Turkey.     

Elimination of tomato mosaic virus by composting tomato residues

Tomato plants infected by tomato mosaic virus (ToMV) were composted. Although the temperature in the stack of tomato residues did not exceed 47°C during composting, ToMV was not detected in samples during composting. Moreover, tomato seedlings grown in pots containing a mixture of Russian peat and the composted material did not become infected. Because ToMV was detected in samples stored in an incubator at 47°C up to the 75th day of the incubation, it was concluded that the biological degradation of ToMV seems to play a more important role than the heat inactivation of the virus.Samenvatting Met tomatemozaïekvirus (ToMV) geïnfecteerde tomateplanten werden gecomposteerd. Hoewel de temperatuur in de composthoop tijdens de compostering niet hoger werd dan 47°C, kon ToMV niet worden geïsoleerd uit monsters, die tijdens de compostering werden genomen. Bovendien werden tomatezaailingen, die in een mengsel van potgrond met gecomposteerd materiaal waren opgegroeid, niet met het virus geïnfecteerd. Omdat ToMV tot 75 dagen na het begin van de incubatie bij 47°C in de monsters kon worden aangetoond, kan worden geconcludeerd, dat de biologische afbraak van ToMV een belangrijker rol speelt dan de inactivering ten gevolge van de warmteontwikkeling.     

ROS and NO production in compatible and incompatible tomato-<Emphasis Type="Italic">Meloidogyne incognita</Emphasis> interactions

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomato-nematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H₂O₂ cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.     

Behavior and mutation of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in <Emphasis Type="Italic">Solanum toxicarium</Emphasis> grown in aseptic culture

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.