Adverse effect of the chitinolytic enzyme PjCHI‐1 in transgenic tomato on egg mass production and embryonic development of Meloidogyne incognita

A novel chitinase gene (PjCHI‐1) isolated from Paecilomyces javanicus, a non‐nematophagous fungus, and driven by a CaMV35S promoter, was delivered into CLN2468D, a heat‐tolerant cultivar of tomato (Solanum lycopersicum). T₁ tomato plants exhibited high endochitinase activity and reduced numbers of eggs and egg masses when infected with the root‐knot nematode (RKN) Meloidogyne incognita. The eggs found in transgenic tomato had lower shell chitin contents than eggs collected from control plants. Egg masses from transgenic plants exhibited higher chitinase activity than those from control plants. Moreover, only 30% of eggs from transgenic plants were able to develop to the multi‐cell/J1 stage, compared with more than 96% from control plants. The present study demonstrated that the expression of the PjCHI‐1 chitinase gene can effectively reduce the production of egg masses and repress the embryonic development of M. incognita, presenting the possibility of a novel agro‐biotechnological strategy for preventing crop damage by RKN.     

Virulence development and genetic polymorphism in Meloidogyne incognita (Kofoid & White) Chitwood after prolonged exposure to sublethal concentrations of nematicides and continuous growing of resistant tomato cultivars

BACKGROUND: The root‐knot nematode, Meloidogyne incognita (Kofoid & White) Chitwood, is an important plant pathogen damaging to tomato. Continuous use of resistant tomato cultivars and nematicides for its effective management might lead to resistance break‐up or nematicide failure. Genetic variability and virulence in M. incognita on susceptible Pusa Ruby tomato were analysed by bioassay, esterase and DNA polymorphism after a 5 year weekly exposure to carbofuran, carbosulfan, cadusafos and triazophos at 0.0125, 0.0250 and 0.0500 µg g^?1. Virulence in M. incognita after a 5 year multiplication on resistant tomatoes was assessed. RESULTS: The nematicidal treatments resulted in the development of virulent M. incognita populations. Their invasion potential increased significantly after continuous exposure to low concentrations of the nematicides. Also, growing resistant tomato cultivars for ten successive seasons resulted in a 6.6% increase in the invasion potential. These virulent populations exhibited 1–3 additional esterase and DNA bands compared with untreated populations. CONCLUSION: A 5 year exposure of M. incognita to sublethal concentrations of nematicides or resistant tomato cultivars exerted enough selection pressure to cause genomic alterations for virulence development. Isozyme markers can be used for rapid and precise diagnostics of field populations by advisory services, enabling judicious remedial management decisions. Copyright © 2009 Society of Chemical Industry     

Screening Tomato Cultivars for Resistance to Meloidogyne Species

Abstract

Screening tests on 162 tomato cultivars and hybrids conducted at the Division of Horticulture, Indian Agricultural Research Institute, New Delhi during 1967-8 indicated a high degree of resistance to root-knot nematodes in Nematex, VFN-8, 65N215-1, 65N255-1 and S1-120, under field and laboratory conditions. The degree of resistance varied with the species of Meloidogyne. Nematex was immune to M. javanica, M. incognita and M. arenaria. VFN-8, 65N215-1 and 65N255-1 were immune to M. incognita and M. arenaria and resistant to M. javanica. S1-120, a commercial cultivar, showed a high degree of tolerance to M. javanica, M. incognita and M. arenaria. The degree of resistance varied with nematode population density.     

The influence of the nematode Meloidogyne incognita on competition between Solanum nigrum and tomato

The effect of Meloidogyne incognita (Kofoid & White) Chitwood, 1949, on the competitive relationship between tomato (Lycopersicon esculentum Mill.) and black nightshade (Solanum nigrum L.) was investigated under glasshouse conditions. Two intraspecific competition treatments were set up for the crop and the weed, and five interspecific treatments where the emergence of S. nigrum plants was progressively delayed in relation to tomato. Nematodes reproduced in all inoculated plants, their multiplication rates being much higher in tomato than in S. nigrum plants. Under nematode-free conditions, intraspecific competition of tomato was more severe than the interspecific competition shown by the weed S. nigrum. Tomato was as tronger competition with S. nigrum than the weed was with itself. However, when infested by M. incognita, both species displayed a similar competitive ability. Tomato yield losses increased with prolonged weed competition but were greater under nematode-infested conditions.     

Host-parasite relationships in tobacco plants infected with a root-knot nematode (<Emphasis Type="Italic">Meloidogyne incognita</Emphasis>) population from the Azores

During a nematode survey, severe infections of tobacco feeder roots and heavy soil infestations byMeloidogyne incognita race 1 were found in S. Miguel (Azores islands, Portugal). This is the first record ofM. incognita infection of tobacco in Azores. Morphology of various life stages, analysis of the esterase electrophoretic pattern and differential host tests were used for nematode characterization and identification. Nematode-induced mature galls were spherical and/or ellipsoidal and usually contained more than one female, males and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. Giant cell cytoplasm was aggregated along a thickened cell wall. Vascular tissues within galls appeared disorganized. The relationship between the initial nematode population density and growth of tobacco plants was tested in a glasshouse experiment in which inoculum levels varied from 0 to 512 eggs and juveniles (J₂) cm⁻³ of soil. Seinhorst’s model was fitted to height and top fresh weight data of the inoculated and control plants. Tolerance limits with respect to plant height and fresh top weight of tobacco cv. ‘Erzegovina’ plants toM. incognita race 1 were estimated as 1.25 eggs and J₂ cm⁻³ of soil. The maximum nematode reproduction rate was 404.7 at an initial population density of 4 eggs and J₂ cm⁻³ of soil. http://www.phytoparasitica.org posting March 2, 2004.     

Histological study of giant cells formed by the root-knot nematodeMeloidogyne artiellia as compared withM. hapla andM. javanica in cabbage, turnip and barley

Feeding sites induced by the root-knot nematodeMeloidogyne artiellia in turnip (Brassica rapa), cabbage (Brassica oleracea) and barley (Hordeum vulgare) were examined by light and electron transmission microscopy, and compared with those formed byM. javanica andM. hapla. The feeding cells ofM. artiellia in turnip and cabbage showed hypertrophy, hyperplasia and vacuolization, and became multinucleate giant cells. In contrast to the typical simple-shaped giant cells ofM. javanica andM. hapla, those ofM. artiellia had an amoeboid structure containing ‘protuberances’, which are distinct from previously reported ‘projects’ of giant cells induced byM. incognita in other plants. Protuberances expanding between vascular cells were observed in young and developed giant cells. Since no cell-wall fragments were found at the bases of the protuberances, either by light or electron microscopy, it is strongly indicated that these structures were caused by local expansion of the giant cells rather than by fusion with adjacent cells. The giant cells ofM. artiellia in barley had regular shapes of giant cells without protuberances, and resembled those induced byM. javanica orM. hapla in turnip and cabbage. http://www.phytoparasitica.org posting Sept. 17, 2006.     

南方根结线虫中国分离群体种内变异分析

为调查我国不同地区和不同寄主上的根结线虫Meloidogyne spp.种类分布以及群体变异情况,基于酯酶和苹果酸脱氢酶同工酶图谱及SCAR分子标记技术对2017—2019年从6省19种植物根部组织分离到的40个根结线虫群体进行鉴定,针对南方根结线虫M.incognita群体分别通过寄主鉴别法进行生理小种鉴别,利用携带Mi抗性基因的番茄进行毒力测试,对2龄幼虫的口针长度和体长进行测量,并对核糖体ITS和线粒体Nad5基因序列进行比较分析。结果显示：根结线虫分离群体经鉴定包括38个南方根结线虫群体和2个象耳豆根结线虫M.erterolobii群体;38个南方根结线虫群体中有35个群体被鉴别为1号生理小种,其余3个群体被鉴别为2号生理小种;发现1个南方根结线虫群体CN19可在携带Mi抗性基因的番茄上侵染繁殖,为毒性群体,其余群体无法进行侵染和繁殖,为无毒群体。南方根结线虫群体2龄幼虫的口针长度和体长均差异较大,而不同寄主来源分离群体的ITS和Nad5基因序列也存在一定变异。基于ITS和Nad5基因序列构建的系统发育树将所有根结线虫群体归为南方根结线虫和象耳豆根结线虫组成的2个独立分支,但不能确定南方根结线虫不同群体的分子进化与其寄主来源和地理分布之间的相关性。     

Resistance to <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> expresses a hypersensitive-like response in <Emphasis Type="Italic">Coffea arabica</Emphasis>

Root-knot nematodes (RKN) are obligate parasite species of the genus Meloidogyne that cause great losses in Arabica coffee (Coffea arabica L.) plantations. Identification of resistant genotypes would facilitate the improvement of coffee varieties aiming at an environmental friendly and costless nematode control. In this work, the C. arabica genotype ‘UFV 408-28’ was found to be resistant to the most destructive RKN species M. incognita. Pathogenicity assays indicated that the highly aggressive populations of M. incognita races 1, 2 and 3 were not able to successfully reproduce on ‘UFV 408-28’ roots and displayed a low gall index (GI = 2). An average reduction of 87% reduction of the M. incognita population was observed on ‘UFV 408-28’ when compared to the susceptible cultivar ‘IAC 15’. By contrast, ‘UFV 408-28’ was susceptible to the related species M. exigua and M. paranaensis (GI = 5 and 4, respectively). Histological observations performed on sections of UFV408-28 roots infected with M. incognita race 1 showed that nematode infection could be blocked right after penetration or during migration and establishment stages, at 6 days, 7 days and 8 days after infection (DAI). Fluorescence and bright field microscopy observations showed that root cells surrounding the nematodes exhibited HR-like features such as accumulation of phenolic compounds and a necrotic cell aspect. In the susceptible ‘IAC 15’ roots, 6 DAI, feeding sites contained giant cells with a dense cytoplasm. Necrotic cells were never observed throughout the entire infection cycle. The HR-like phenotype observed in the ‘UFV 408-28’—M. incognita interaction suggests that the coffee resistance may be mediated by a R-gene based immunity system and may therefore provide new insights for understanding the molecular basis of RKN resistance in perennial crops.     

Interaction of Root-knot Nematodes (RKN) and the Bacterium Agrobacterium Tumefaciens in Roots of Prunus Cerasifera: Evidence of the Protective Effect of the Ma RKN Resistance Genes Against Expression of Crown Gall Symptoms

Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.     

Suppression of Meloidogyne incognita by the endophytic fungus Acremonium implicatum from tomato root galls

Specific endophytes with biocontrol potential might occur in diseased plant tissues. We isolated an endophytic fungus from tomato root galls infected with Meloidogyne incognita and identified it as Acremonium implicatum based on morphology and internal transcribed spacer sequences. Its biocontrol potential was tested in vitro and in pot and field experiments. In the in vitro test, 96.0% of second-stage juveniles of M. incognita were killed by a culture filtrate of A. implicatum after 48 h. The fungus also suppressed egg hatching, with only 36.3% of treated eggs hatching compared with 87.3% of control eggs. Pot experiments showed that A. implicatum inhibited the formation of root galls, with 40.6 galls per treated plant compared with 121.6 on control plants. A. implicatum reduced the nematode population in soil, with 151.1 nematodes per 100 g treated soil and 375.1 in control soil. Field experiments demonstrated that the root gall index of treated plants (25) was markedly lower than that of control plants (96). In conclusion, A. implicatum has excellent potential for the biocontrol of M. incognita.     

Pathogenicity and host‐parasite relationships of the root‐knot nematode Meloidogyne incognita on celery

Pathogenicity and host‐parasite relationships in root‐knot disease of celery (Apium graveolens ) caused by Meloidogyne incognita race 1 were studied under glasshouse conditions. Naturally and artificially infected celery cv. D’elne plants showed severe yellowing and stunting, with heavily deformed and damaged root systems. Nematode‐induced mature galls were spherical and/or ellipsoidal and commonly contained more than one female, males and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. The cytoplasm of giant cells was aggregated along their thickened cell walls and consequently the vascular tissues within galls appeared disrupted and disorganized. The relationship between initial nematode population density (Pi) and growth of celery plants was tested in glasshouse experiments with inoculum levels that varied from 0 to 512 eggs and second‐stage juveniles (J₂) mL^?1 soil. Seinhorst's model y = m + (1 – m)z^P–T was fitted to height and top fresh weight data of the inoculated and control plants. The tolerance limit with respect to plant height and fresh top weight of celery to M. incognita race 1 was estimated as 0·15 eggs and J₂ mL^?1 soil. The minimum relative values (m) for plant height and top fresh weight were 0·37 and 0·35, respectively, at Pi ≥ 16 eggs and J₂ mL^?1 soil. The maximum nematode reproduction rate (Pf/Pi) was 407·6 at an initial population density (Pi) of 4 eggs and J₂ mL^?1 soil.     

Stimulation of respiratory pathways in tomato roots infested by Meloidogyne incognita

Respiration of tomato roots susceptible and resistant to Meloidogyne incognita was measured during infestation. No significant changes in respiratory rate occurred in susceptible tomato roots, during infestation by M. incognita. In resistant tomato roots, a pronounced increase of both cyanide-sensitive and cyanide-resistant oxidases, was observed during nematode attack. The time-course of the respiration during 12 days, after nematode inoculation, showed that resistant tomato roots responded with a rapid increase in cyanide-sensitive and cyanide-resistant respiration as invading nematodes progressed; no changes were observed in the susceptible tomato roots.Change in the rate of oxygen uptake paralleled an increase in nematode density in resistant tomato roots; oxygen uptake rose linearly to an infestation level of 50 juveniles for each seedling, above which value it declined. The physiological significance of the alternative respiratory pathway is discussed.     

Occurrence of Resistance-breaking Populations of Root-knot Nematodes on Tomato in Greece

Nine populations of Meloidogyne spp. from Greece have been identified as M. javanica or M. incognita using either isozyme phenotypes or the sequence characterized amplified region-polymerase chain reaction (SCAR-PCR) technique. Virulence against the Mi resistance gene was assayed by pot experiments in controlled conditions and revealed the ability of five populations of M. javanica and one population of M. incognita to reproduce on tomato cultivars containing that gene. A resistance-breaking population of M. incognita is reported for the first time in the country; the M. javanica populations constitute new records for the Greek mainland.     

Analysis of the expression patterns of the Arabidopsis thaliana tubulin-1 and Zea mays ubiquitin-1 promoters in rice plants in association with nematode infection

The temporal and spatial expression of three promoters was investigated in transgenic rice plants using promoter-β-glucuronidase (gusA) reporter gene fusions. The promoters studied were ubiquitin-1 (UBI-1) of Zea mays, Cauliflower Mosaic Virus 35S gene (CaMV35S) and a tubulin gene (TUB-1) of Arabidopsis thaliana. The TUB-1 promoter provided 7.32-fold more GUS activity in roots relative to tillers. This was significantly different from the corresponding value of 2.82-fold for CaMV35S but not from that of 4.55-fold for UBI-1, activity of both promoters was higher in the root tips and zone of elongation than mature roots. This younger root tissue represented a declining proportion of the expanding root system with time. Older tissue expressing GUS under control of the TUB-1 promoter showed a steeper decline in activity with time than occurred with the UBI-1 promoter. Nematode infection did not alter the overall pattern of expression from the two promoters, except that the giant cells induced by Meloidogyne incognita retained TUB-1 promoter activity as roots matured. Pratylenchus zeae invaded older root regions than M. incognita and no changes in promoter activity were detected where it fed. The results suggest the TUB-1 promoter has characteristics that favour its use for delivering anti-feedants, such as cysteine proteinase inhibitors, to M. incognita.     

Screening tomato genotypes for resistance and tolerance to Tomato chlorosis virus

Tomato chlorosis virus (ToCV) is an emerging crinivirus in Brazil that causes an economically important disease in tomato (Solanum lycopersicum) and other solanaceous species. ToCV is transmitted predominantly by the whitefly Bemisia tabaci Middle East‐Asia Minor 1 (MEAM1, formerly biotype B), in a semipersistent manner. As all cultivated tomato varieties and hybrids are susceptible to this crinivirus, the main alternatives for the control of the disease are the use of healthy seedlings for transplanting and the chemical control of the insect vector. The objective of this work was to evaluate the responses of tomato genotypes to infection with this crinivirus and their tolerance to the disease in order to support the development of other alternatives for disease control. Resistance to infection was evaluated by ToCV inoculation with viruliferous B. tabaciMEAM1 followed by virus detection by RT‐PCR and RT‐qPCR. To measure tolerance to the disease, plant development and fruit yield of ToCV‐infected and healthy plants were compared. Among 56 genotypes, only the lineage IAC‐CN‐RT (S. lycopersicum ‘Angela Gigante’ × S. peruvianum ‘LA 444‐1’) was highly resistant to infection with ToCV. Tolerance to the disease over two trials with different genotypes showed variable results. The effect of ToCV on plant development varied from 2.9% to 71.9% reduction, while yield loss varied from 0.2% to 51.8%. The highly ToCV‐resistant lineage IAC‐CN‐RT, which is also resistant to a Spanish isolate of ToCV, might be useful for tomato breeding programmes.     

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.     

三种化合物处理对南方根结线虫生长发育及致病能力的影响

为明确不同浓度NH4HCO3、Cu SO4·5H2O和Fe Cl3·6H2O对南方根结线虫Meloidogyne incognita 2龄幼虫活性的影响,在盆栽条件下将化合物处理后的2龄幼虫接种番茄,通过根染色和根系冰冻切片法观察2龄幼虫的发育状况和根系内巨细胞的形成,并测定了番茄根系各生理指标的变化。结果表明,50mmol/L NH4HCO3、1.4mmol/L Cu SO4·5H2O和1.4 mmol/L Fe Cl3·6H2O处理南方根结线虫后,番茄根系内2龄、3龄及4龄幼虫数量均显著小于对照;根系内丙二醛、可溶性糖和游离氨基酸的含量均显著低于对照,根系活力显著增强;经NH4HCO3处理后,根系内巨细胞形成缓慢,14 d时出现巨型细胞空洞现象。3种化合物处理对南方根结线虫生长发育的抑制作用显著,处理后的线虫对番茄植株致病力减弱,表现为NH4HCO3抑制作用最强,Cu SO4·5H2O次之,Fe Cl3·6H2O最弱。     

Nematicidal Activity of Chrysanthemum coronarium

Organic amendments and green manure are potential alternatives to the harmful chemical control means currently used against plant-parasitic nematodes. In this work, Chrysanthemum coronarium was applied to the soil as a green manure to control the root-knot nematodes Meloidogyne incognita and M. javanica. Chrysanthemum coronarium significantly reduced nematode infection of tomato roots and improved plant-top fresh weight, both in the greenhouse and in microplots. Other green manures, derived from Anthemis pseudocotula, wild chickpea (Cicer pinnatifidum), Geranium spp. and wheat, were not as effective as C. coronarium. Chrysanthemum coronarium, retained its nematicidal activity even when applied as a dried material. Only mature C. coronarium plants, in their flowering stage, exhibited nematode control activity, but the green plant parts were more effective than the flowers. An aqueous extract of C. coronarium exhibited in vitro, nematostatic activity towards M. incognita and M. javanica second-stage juveniles and inhibited their hatching from eggs and egg-masses; its nematostatic activity was expressed also against other phytonematode species such as Heterodera avenae and Pratylenchus mediterraneus, but did not affect the beneficial entomopathogenic nematode Steinernema feltiae.