Sergio Molinari

Expression of tomato salicylic acid (SA)-responsive pathogenesis-related genes in Mi-1-mediated and SA-induced resistance to root-knot nematodes.

The expression pattern of pathogenesis-related genes PR-1, PR-2 and PR-5, considered as markers for salicylic acid (SA)-dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre-treated with SA and subsequently infected with root-knot nematodes (RKNs) (Meloidogyne incognita). PR-1 was up-regulated in both roots and shoots of SA-treated plants, whereas the expression of PR-5 was enhanced only in roots. The over-expression of PR-1 in the whole plant occurred as soon as 1 day after SA treatment. Up-regulation of the PR-1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA-treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR-1, PR-2 and PR-5 was also examined in the roots and shoots of susceptible and Mi-1-carrying resistant tomato plants infected by RKNs. Nematode infection produced a down-regulation of PR genes in both roots and shoots of SA-treated and untreated plants, and in roots of Mi-carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR-1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR-like response to RKNs in tomato.

Natural genetic and induced plant resistance, as a control strategy to plant-parasitic nematodes alternative to pesticides

Plant-parasitic nematodes are pests of a wide range of economically important crops, causing severe losses to agriculture. Natural genetic resistance of plants is expected to be a valid solution of the many problems nematodes cause all over the world. Progress in resistance applications is particularly important for the less-developed countries of tropical and subtropical regions, since use of resistant cultivars may be the only possible and economically feasible control strategy in those farming systems. Resistance is being considered of particular importance also in modern high-input production systems of developed countries, as the customary reliance on chemical nematicides has been restricted or has come to an end. This review briefly describes the genetic bases of resistance to nematodes in plants and focuses on the chances and problems of its exploitation as a key element in an integrated management program. Much space is dedicated to the major problem of resistance durability, in that the intensive use of resistant cultivars is likely to increasingly induce the selection of virulent populations able to “break” the resistance. Protocols of pest-host suitability are described, as bioassays are being used to evaluate local nematode populations in their potential to be selected on resistant germplasm and endanger resistant crops. The recent progress in using robust and durable resistances against nematodes as an efficient method for growers in vegetable cropping systems is reported, as well as the possible use of chemicals that do not show any unfavorable impact on environment, to induce in plants resistance against plant-parasitic nematodes.

New developments in understanding the role of salicylic acid in plant defence

AbstractSalicylic acid (SA) is currently considered as a key molecule in activating plant defence againstvarious insects and pathogenic invaders. Its role in determining the so-called systemic acquiredresistance (SAR), that confers long-lasting protection against a broad spectrum of pathogens, hasdefinitely been established. The activation of an SA-dependent signalling pathway is also associatedwith more specific plant defences, such as those determined by gene-for-gene recognition of thepathogen by plants carrying resistance genes (R-genes). A recent further impulse in the investi-gation of the role of SA, both in local defences and SAR, has come either from genetic screens ofArabidopsis or from SA-degrading transgenic plants that express the bacterial salicylate hydroxylasegene (NahG). Genetic screens of Arabidopsis and the recently developed microarray technologyallowed us to identify several genes involved in SA synthesis and signalling. However, the bio-chemical mechanisms by which SA can exert its many functions are not clear yet and the scope ofthis review is to report on such mechanisms. SA signalling is indicated here as a complex networkoperating through gene expressions and biochemical pathways which are finely regulated by SAconcentration. According to its level in plants, SA may operate as a beneficial anti-inflammatorycompound or as a cell killer. New developments in understanding the role of SA in plant defenceare reported from studies on root response to soil pests, such as root-knot nematodes. The roleof SA in diseases of roots has been reviewed here for the first time, as most of our knowledge onthis matter is based on studies carried out on leaves.Keywords: Salicylic acid, Plant resistance, Root diseases, Hypersensitive reaction, Reactive oxygen species,Programmed cell death

Systemic acquired resistance activation in solanaceous crops as a management strategy against root-knot nematodes

BACKGROUND Activators of systemic acquired resistance (SAR), such as salicylic acid (SA) and its synthetic functional analogues benzo(1,2,3)thiadiazole-7-carbothionic acid-S-methyl ester (BTH) and 2,6-dichloroisonicotinic acid (INA), were tested on tomato, eggplant and pepper for the control of the root-knot nematode Meloidogyne incognita. Effects on plant fitness, nematode reproduction and root galling were screened in relation to different methods of application, to different applied dosages of chemicals and to different plant growth stages. Dosages applied to plants were in relation to plant weights. These chemicals were also tested for their possible nematotoxic activity in vitro. RESULTS Soil drenches of SA and INA and root dip application of SA and BTH inhibited nematode reproduction, at specific dosage ranges, without affecting plant growth. SA and INA were able to reduce root galling as well. Foliar sprays of both SA and BTH were ineffective against nematode attacks. Plants tolerated SA more than the other chemicals tested. BTH at elevated concentrations increased the mortality of nematode juveniles and reduced egg hatching in vitro. CONCLUSIONS SAR activators at concentrations suitable for different plant growth stages and applied by the proper method can possibly be included in IPM programmes for nematode management.

Antioxidant enzymes in (a)virulent populations of root-knot nematodes.

Assays of antioxidant enzymes, including catalase, peroxidase and superoxide dismutase (SOD), have been carried out on extracts of females and second-stage juveniles (J2) of a pair of Meloidogyne incognita isolates, one virulent and one avirulent, selected on tomato, and an avirulent field population of M. incognita. Catalase and SOD activity were found to be higher in extracts of the virulent isolate SM1 when compared with the avirulent counterparts. Peroxidase activity, assayed with o-dianisidine as the substrate, was enhanced in SM1 J2 compared with the avirulent avr1 J2. Catalase isozymes were separated by isoelectrofocusing into a very acidic and a basic isoform; this latter isoform was found to be responsible for the enhancement of catalase activity in virulent populations. SOD isozyme electrophoresis patterns (IEP) of root-knot nematodes, obtained by native PAGE, showed the presence of slow- and fast-migrating bands. SOD IEP of virulent females contained a slow-migrating band with a relative mobility (Rm) on the gels slightly higher (0.52) than the corresponding band from avirulent populations (0.50). This change was confirmed with native PAGE gels loaded with extracts from J2. To check how widespread this change is in field populations of RKN, a survey of SOD IEP from 12 RKN field (a)virulent populations was carried out. The specificity of the 0.52 Rm band for virulent populations was confirmed. Separation by native PAGE of peroxidases, stained either by o-dianisidine or diamino-benzidine, showed two isoforms with no apparent differences between the populations tested.

SOD polymorphism in the Xiphinema americanum -group (Nematoda: Longidoridae)

Isoelectrofocusing of superoxide dismutase (SOD) isoforms was carried out on the extracts of 117 nematode populations belonging to the so-called Xiphinema americanum -group. These populations came from the USA (77), Chile (5), Argentina (1), Venezuela (5), Portugal (15), Italy (2), Crete (1), Montenegro (1), Slovakia (4), Hungary (3), Egypt (1) and India (2). A total of 17 bands of enzyme activity were observed in the screening, whilst single enzyme phenotypes showed from two to eight bands. The high degree of SOD polymorphism of this nematode collection was grouped by cluster analysis into seven distinct homogeneous groups characterised by specific combinations of SOD markers. Sub-groups could be discriminated for larger groups. The small Groups 3 and 5 were constituted mostly by populations from USA east coast states ( i.e ., NY and PA, respectively). The larger Group 1 resulted from the association of populations coming from various and distant North American States. In other large groups, North American populations were associated with South American and European populations. Overall, the data presented here suggest that geographic separation and different hosts do not seem to be the source of genetic diversity for the X. americanum -group. When an adequate number of populations were collected from the same country, the variability expressed by such a sub-sample was comparable to that of the whole nematode collection. For the first time, homogeneous populations of a large collection of X. americanum -group populations were associated by molecular means in order to explore further approaches that may help resolve the recalcitrant taxonomy and phylogeny of this much debated group.

Superoxide dismutase isoelectric focusing patterns as a tool to differentiate pathotypes of Globodera spp.

Isoelectric focusing was used to separate proteins from cyst extracts of potato cyst nematode (PCN) populations. In a first set of assays, cyst extracts from standard populations of Globodera rostochiensis pathotypes Ro1, Ro2, Ro3, Ro2/3, Ro4, and Ro5, and G. pallida pathotypes Pa2 and Pa3, were loaded on isoelectric focusing gels. Gels were stained for superoxide dismutase (SOD), esterase, and glucose-6-phosphate isomerase (GPI). Twelve bands of SOD activity were detected, six (B1-B6) migrating towards the basic zone and the other six (A1-A6) migrating towards the acidic zone, starting from the loading point. A cluster analysis was carried out based on a data matrix that reported the presence or absence of SOD bands on the isozyme electrophoresis patterns (IEPs). Globodera spp. were clearly distinguished and, within G. rostochiensis , Ro2 and Ro4 shared a high level of similarity, respectively, with Ro3 and Ro5; moreover, Ro1 could be clearly distinguished from Ro2/3 and Ro4/5. Globodera pallida Pa2 and Pa3 also shared a high level of similarity. In contrast, esterase and GPI IEPs did not discriminate among G. rostochiensis standard pathotypes. Subsequently, 14 field populations of G. rostochiensis , five from Italy and nine from Venezuela, and three field populations of G. pallida , two from Italy and one from Chile, were assayed to obtain SOD IEPs. Italian populations had previously been identified at pathotype level by bioassays according to the generally accepted international test using different resistant potato cultivars and clones. The cluster analysis carried out on the SOD IEPs of all the populations tested formed four distinct groups within G. rostochiensis and only one within G. pallida . Pathotype identification of Globodera populations by SOD IEPs was not able to discriminate between bioassay standard couples Ro2/Ro3, Ro4/Ro5 and Pa2/Pa3. Therefore, three groups were assigned to Ro1, Ro2/3 and Ro4/5, and a fourth group to Pa2/Pa3. Four Venezuelan populations, not identified at pathotype level by bioassays, formed a distinct fifth group. By means of the method described herein, four additional unknown Venezuelan populations could be assigned to Ro1 group and one to Ro2/Ro3 group; one G. pallida population from Chile was assigned to Pa2/Pa3 group.

Superoxide Dismutase as a Tool for the Mulacular Identification of Plant Parasitic Nematodes.

Superoxide dismutase (SOD) is a constitutive family of enzymes produced by all aerobic organisms. Varying amounts of SOD activity have been found at all life stages of the most diffused plant parasitic nematodes. SOD is important to aerobic metabolism and parasitism of nematodes in that it catalyzes the first step of the neutralization of the highly toxic superoxide anion (O2 •-), which is largely produced in plant-nematode incompatible reactions. SOD has also been shown to be a significant tool to diagnose root-knot, cyst-, and longidorid nematodes. A high SOD polymorphism has been revealed by Native-Page on gradient polyacrylamide gels for Meloidogyne spp. and by isoelectrofocusing for Globodera, Xiphinema and Longidorus spp. The sensitivity of such procedures has been improved by using the PhastSystem (Amersham Biosciences, Piscata, NJ, USA), an automated equipment for electrophoresis. An accurate discrimination of species of all the nematode genera tested has been achieved and an attempt was made to group populations of the Xiphinema americanum-group and to detect Globodera rostochiensis and G. pallida pathotypes.

Manganese superoxide dismutase in Meloidogyne incognita isolates selected for virulence on Mi-1-carrying tomato: gene expression and enzyme activity

Root knot nematodes (Meloidogyne spp.) are important pests of a wide range of crops, including tomato. Resistance of tomato to root-knot nematodes is conferred by the single dominant gene Mi-1, which currently is present in all commercially available resistant tomato cultivars. However, several resistance-breaking populations are being collected worldwide. Two isolates coming from the same standard population of Meloidogyne incognita, one selected for virulence against Mi-1 (SM1) and the other left avirulent (avr1), have been used in this study. qRT-PCR was used to detect transcript levels of the manganese superoxide dismutase (Mn-SOD) gene from (a)virulent pre-parasitic second-stage juveniles (J2). Over-expression of the Mn-SOD gene was found in the virulent isolate compared with the avirulent counterpart. The enzyme activity of membrane-bound mitochondrial Mn-SOD was assayed in J2 and adult females as the fraction of total SOD activity insensitive to hydrogen peroxide (H2O2). J2 from SM1 showed about a two-fold higher enzyme activity than J2 from avr1; conversely, no difference was found when adult females were tested. Proteins of J2 extracts were separated by n-PAGE on special mini-gels and stained for SOD. One slow migrating and three fast migrating bands were stained. SOD activity of the slow migrating band was H2O2-insensitive and enriched by treatment with the detergent Triton X-100. J2 survival was monitored in suspensions provided or not provided with the cell oxygen radical generator paraquat, at high concentration. Virulent J2 responded to paraquat treatment by increasing life extension with respect to control conditions; by contrast, avirulent J2 suffered major mortality in the presence of paraquat. Mn-SOD gene expression is discussed in relation to nematode fitness in oxidative stresses and vir phenotype.