Optimisation of culture conditions for in vitro infection of tomato with the root-knot nematode Meloidogyne javanica

Conditions for in vitro culture of tomato seedlings (Lycopersicon esculentum) were optimised to yield high in vitro rates of infection by the root-knot nematode Meloidogyne javanica. The influence of nutrient salts, gelling agents, sucrose concentration and pH on the development of nematode-induced root galis was investigated. A quarter-strength Murashige and Skoog medium supplemented by 0.5% sucrose and solidified with 0.6% phytagel at pH 6.4 gave most galis on tomato roots. Eggs were sterilised in 2.5% sodium hypochlorite for 4 min followed by 0.2% mercuric chloride for 4 min. Surface-sterilised eggs showed a 16% cumulative hatching rate within 10 days. Tomato seedlings cultivated in vitro for 1 week were inoculated with sterile eggs and the infection process was monitored weekly. After 5 to 7 weeks, sterile egg masses were harvested, second-stage juveniles were hatched in sterile distilled water and used to re-inoculate tomato seedlings without further sterilisation. The culture conditions described gave similar infection results for the related root-knot nematodes M. incognita, M. arenaria and M. hapla

Auxin induction is a trigger for root gall formation caused by root-knot nematodes in white clover and is associated with the activation of the flavonoid pathway

We studied the expression of the auxin responsive promoter (GH3) fused to the gusA reporter gene in white clover (Trifolium repens cv. Haifa) during the initiation of root galls by root-knot nematodes (Meloidogyne javanica) to investigate whether nematode infection affects auxin distribution in developing galls. In search for a plant signal that would mediate changes in auxin location we studied the induction of the flavonoid pathway because flavonoids can act as auxin transport regulators. Three chalcone synthase (CHS1, CHS2 and CHS3) promoter:gusA fusions were examined in transgenic plants and flavonoids were detected using fluorescence microscopy. Within 24 h post inoculation CHS:gusA expression occurred around the invading nematode. At 48 h post inoculation CHS:gusA expression and flavonoids were detected throughout the infection site, followed by high GH3:gusA expression in the gall 48–72 h post inoculation. Initially (48–72 h post inoculation) high GH3:gusA expression in giant cell precursors was followed by low expression in the enlarging giant cells (96–120 h post inoculation), suggesting that auxin is needed as a trigger for giant cell initiation but not for later enlargement. We suggest that nematodes control auxin distribution in the root and that flavonoids could be responsible for controlling auxin accumulation

Influence of the erineum strain of Colomerus vitis (Acari: Eriophyidae) on grape (Vitis vinifera) defense mechanisms

Grape (Vitis vinifera) is commonly affected by the erineum strain of Colomerus vitis (GEM) in Iran and the susceptibility of grape cultivars to GEM is poorly understood. In order to evaluate the impact of GEM on grape and its defense mechanisms against the mite, an exploratory study was carried out on 19 cultivars (18 Iranian and the non-native Muscat Gordo). The differential susceptibility of cultivars to GEM was compared on the basis of the area of leaf damage induced by GEM. The cultivars White Thompson seedless of Bovanat, Atabaki Zarghan, Koladari Ghoochan and Sahebi Uroomie were less susceptible to GEM, whereas Ghalati Dodaj, Rishbaba, Muscat Gordo and Neyshaboori Birjand appeared to be the most affected by the mite. In a no-choice setup, plants of selected cultivars of these two groups were infested by GEM and assayed for 10 biomarkers usually related to plant stress mechanisms against plant feeders: the activity of defense enzymes—peroxidase (POX), polyphenol oxidase (PPO), superoxide dismutase (SOD), phenylalanine ammonia-lyase (PAL), catalase (CAT), the amount of total polyphenolics, total flavonoids, total soluble carbohydrates, hydrogen peroxide (H2O2), and malondialdehyde (MDA) expressing lipid peroxidation. The biomarkers were assessed in grape leaves 7 days before releasing the mites, as well as 7, 14 and 28 days after infestation (DAI). The activity of the enzymes and the amount of the compounds usually increased in percentage after mite infestation. A significant negative correlation was found between the area of leaf damage and PPO, POX, SOD, MDA and H2O2for all sampling dates. The area of leaf damage showed a significant positive correlation with total soluble carbohydrates at 28 DAI, and significant negative correlations with CAT (at 14 and 28 DAI), PAL and total flavonoids (at 7 DAI). No correlation was observed between area of leaf damage and total polyphenolics. The biomarkers PPO, SOD, CAT activity and H2O2provided the best explanation for the response of grape cultivars to GEM infestation