<Emphasis Type="Italic">CaMi</Emphasis>, a root-knot nematode resistance gene from hot pepper (<Emphasis Type="Italic">Capsium annuum</Emphasis> L.) confers nematode resistance in tomato

Several root-knot nematode (Meloidogyne spp.) resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, none of them has yet been cloned. In this study, a candidate root-knot nematode resistance gene (designated as CaMi) was isolated from the resistant pepper line PR 205 by degenerate PCR amplification combined with the RACE technique. Expression profiling analysis revealed that this gene was highly expressed in roots, leaves, and flowers and expressed at a lower level in stems and was not detectable in fruits. To verify the function of CaMi, a sense vector containing the genomic DNA spanning the full coding region of CaMi was constructed and transferred into root-knot nematode susceptible tomato plants. Sixteen transgenic plants carrying one to five copies of T-DNA inserts were generated from two nematode susceptible tomato cultivars. RT-PCR analysis revealed that the expression levels of CaMi gene varied in different transgenic plants. Nematode assays showed that the resistance to root-knot nematodes was significantly improved in some transgenic lines compared to untransformed susceptible plants, and that the resistance was inheritable. Ultrastructure analysis showed that nematodes led to the formation of galls or root knots in the susceptible lines while in the resistant transgenic plants, the CaMi gene triggered a hypersensitive response (HR) as well as many necrotic cells around nematodes. Rugang Chen and Hanxia Li are contributed equally to this work.     

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato

Purpose

Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants.

Methods

Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis.

Results

Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans.

Conclusion

Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras

Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e. plants with genetically different cell populations in different cell layers. The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves. The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors. Southern and northern blot analysis confirmed the chimeric nature of such plants. Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds. The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring. The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.     

Effective delivery of a nematode‐repellent peptide using a root‐cap‐specific promoter

The potential of the MDK4‐20 promoter of Arabidopsis thaliana to direct effective transgenic expression of a secreted nematode‐repellent peptide was investigated. Its expression pattern was studied in both transgenic Arabidopsis and Solanum tuberosum (potato) plants. It directed root‐specific β‐glucuronidase expression in both species that was chiefly localized to cells of the root cap. Use of the fluorescent timer protein dsRED‐E5 established that the MDK4‐20 promoter remains active for longer than the commonly used constitutive promoter CaMV35S in separated potato root border cells. Transgenic Arabidopsis lines that expressed the nematode‐repellent peptide under the control of either AtMDK4‐20 or CaMV35S reduced the establishment of the beet cyst nematode Heterodera schachtii. The best line using the AtMDK4‐20 promoter displayed a level of resistance >80%, comparable to that of lines using the CaMV35S promoter. In transgenic potato plants, 94.9 ± 0.8% resistance to the potato cyst nematode Globodera pallida was achieved using the AtMDK4‐20 promoter, compared with 34.4 ± 8.4% resistance displayed by a line expressing the repellent peptide from the CaMV35S promoter. These results establish the potential of the AtMDK4‐20 promoter to limit expression of a repellent peptide whilst maintaining or even improving the efficacy of the cyst‐nematode defence.     

Chemical activators: A novel and sustainable management approach for Meloidogyne incognita (Kofoid and White) Chitwood in Chamomilla recutita L

Use of chemical activator for the management of root-knot disease in medicinal and aromatic plants can become an attractive alternative to traditionally used nematicides. Large numbers of chemical molecules are present in the plants and are involved in the induction of different types of proteins. The purpose of our research study is to explore the possibilities of resistance factors that are inherent in the plant by treating them with few chemical activators to activate against root-knot nematode infection. Efforts were made to achieve a satisfactory suppression of root-knot nematode, Meloidogyne incognita, a serious menace to successful cultivation of chamomile, Matricaria recutita L. Rauch (syn. Matricaria chamomilla L. Fain. Asteraceae) causing root-knot disease through use of different chemical activators. Here we examined selected groups of resistance activator viz. Isonicotinamide, 2-chloronicotinic acid, 5-nitrosalicylic acid, 4-chlorosalicylic acid, DL-2 aminobutyric acid, 2-aminobutyric acid, O-acetylsalicylic acid, 4-amino salicylic acid, salicylic acid and these were used as soil drench using 3 week old seedlings transplanted to root-knot nematode infested pots. Maximum reduction in root-knot severity and nematode population occurred with 4-chlorosalicylic acid, O-acetyl salicylic acid, 2-chloronicotinic acid and gave significant flower yield advantages. Present experiment suggests a strong possibility of these activators in integrated management for protection against plant parasitic nematodes.     

Interaction of vesicular-arbuscular mycorrhizal fungi and root knot nematode on cultivars of tomato and white clover susceptible to Meloidogyne hapla

The interactive effects of vesicular-arbuscular mycorrhizal (VAM) fungi and root-knot nematode (Meloidogyne hapla) were studied on nematode-susceptible cultivars of tomato (cv. Scoresby) and white clover (cv. Huia) at four levels of applied phosphate. The relative merits of simultaneous inoculation with mycorrhizal fungi and nematodes and of inoculation with mycorrhizal fungi prior to nematode inoculation were evaluated. Mycorrhizal plants were more resistant than non-mycorrhizal plants to root-knot nematode at all phosphate levels and growth benefits were generally greater in plants preinfected with mycorrhizal fungi. Nematode numbers increased with increasing levels of applied phosphate. In mycorrhizal root systems, nematode numbers increased in the lower phosphate soils; at higher phosphate levels nematode numbers were either unaffected or reduced. The numbers of juveniles and adults per gram of root were always lower in mycorrhizal treatments. Mycorrhizal root length remained unaffected by nematode inoculation. Mycorrhizal inoculation thus increased the plants' resistance to infection by M. hapla. This was probably due to some alteration in the physiology of the root system but was not entirely a result of better host nutrition and improved phosphorus uptake by mycorrhizal plants.     

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30???M aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The ¹³C-NMR analysis with NaH¹³CO₃ showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500???M Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.     

Identification of Sources of Resistance to Four Species of Root-knot Nematodes in Tobacco

Resistance to the southern root-knot nematode, Meloidogyne incognita races 1 and 3, has been identified, incorporated, and deployed into commercial cultivars of tobacco, Nicotiana tabacum. Cultivars with resistance to other economically important root-knot nematode species attacking tobacco, M. arenaria, M. hapla, M. javanica, and other host-specific races of M. incognita, are not available in the United States. Twenty-eight tobacco genotypes of diverse origin and two standard cultivars, NC 2326 (susceptible) and Speight G 28 (resistant to M. incognita races 1 and 3), were screened for resistance to eight root-knot nematode populations of North Carolina origin. Based on root gall indices at 8 to 12 weeks after inoculation, all genotypes except NC 2326 and Okinawa were resistant to M. arenaria race 1, and races 1 and 3 of M. incognita. Except for slight root galling, genotypes resistant to M. arenaria race 1 responded similarly to races 1 and 3 of M. incognita. All genotypes except NC 2326, Okinawa, and Speight G 28 showed resistance to M. javanica. Okinawa, while supporting lower reproduction of M. javanica than NC 2326, was rated as moderately susceptible. Tobacco breeding lines 81-R-617A, 81-RL- 2K, SA 1213, SA 1214, SA 1223, and SA 1224 were resistant to M. arenaria race 2, and thus may be used as sources of resistance to this pathogen. No resistance to M. hapla and only moderate resistance to races 2 and 4 of M. incognita were found in any of the tobacco genotypes. Under natural field infestations of M. arenaria race 2, nematode development on resistant tobacco breeding lines 81-RL-2K, SA 1214, and SA 1215 was similar to a susceptible cultivar with some nematicide treatments; however, quantity and quality of yield were inferior compared to K 326 plus nematicides.     

Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses

Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants.     

Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol

A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groudnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.     

Effects of altered citrate synthase and isocitrate dehydrogenase expression on internal citrate concentrations and citrate efflux from tobacco (Nicotiana tabacum L.) roots

To assess the effectiveness of manipulating citrate metabolism with the aim of increasing citrate efflux from roots, we generated transgenic tobacco (Nicotiana tabacum L.) lines that either overexpressed mitochondrial citrate synthase (EC 4.1.3.7) activity or had reduced activity of cytosolic isocitrate dehydrogenase (EC 1.1.1.42). Despite increases in citrate synthase activities in transgenic lines of up to 5-fold, neither internal citrate concentrations nor citrate efflux were increased compared to controls suggesting that, in tobacco, citrate synthase activity does not directly determine citrate accumulation and efflux. Consistent with a lack of effect on citrate efflux, the increase in citrate synthase activity did not enhance the aluminium resistance of the transgenic lines. Preliminary data collected on two transgenic lines with cytosolic isocitrate dehydrogenase activities reduced to one-tenth and one third of the control for shoot and root tissues respectively, showed that while these changes in activities were associated with a 1.5-fold increase in internal citrate concentrations of both types of tissue, citrate efflux from roots was not increased. Further work is needed to establish whether the increase in internal citrate concentration is associated with enhanced aluminium resistance of these lines. We conclude that in tobacco internal citrate concentrations and citrate efflux are largely insensitive to large changes in either mitochondrial citrate synthase or cytosolic isocitrate dehydrogenase activities and suggest that other factors, such as transport out of the roots, control citrate efflux.     

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

Summary The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3 end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.     

Overexpression of sense and antisense ced-9 in tobacco plants confers resistance to Meloidogyne incognita

Transgenic tobacco plants expressing the Caenorhabditis elegans programmed cell death gene ced-9, in both sense and antisense orientations, were produced using Agrobacterium tumefaciens-mediated transformation. The generated transgenic tobacco plants were tested for resistance to the root-knot nematode Meloidogyne incognita by measuring gall formation, size of galls generated, and the ability of juvenile-2 (J2) to hatch. Results showed that expression of ced-9 gene in either sense (ced-9F) or antisense (ced-9R) orientation in hemizygous transgenic tobacco plants induced prevention of M. incognita proliferation (as measured by gall number reduction) and J2 hatching. Furthermore, the results also showed that ced-9R in homozygous transgenic tobacco plants prevented J2 hatching, whereas ced-9F homozygous transgenic tobacco plants lost nematicidal function. Although our study demonstrates that expression of either ced-9R or ced-9F genes in tobacco plants significantly reduces infection by M. incognita, further investigation is required to understand the specific mechanisms involved for this control. It is possible that the nematode resistance seen with both sense (ced-9F) and antisense (ced-9R) sequences is the result of two independent mechanisms, one acting on invading nematodes and the other acting during embryogenesis of M. incognita, ultimately resulting in plant protection.