Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands.

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.

Molecular detection assay of the bud mite Trisetacus juniperinus on Cupressus sempervirens in nurseries of central Italy.

Trisetacus juniperinus (Nalepa) sensu Keifer (Acari: Eriophyoidea: Phytoptidae) causes irregular development of buds, shoot deformations and stunted growth of trees, resulting in a serious threat to nurseries and young stands of Cupressus sempervirens L. (Mediterranean cypress). Recently, some cypress clones selected for their resistance to the fungal canker agent Seiridium cardinale (Wag.) have shown high susceptibility to the mite. Considering its tiny body, its hidden lifestyle inside the buds and the probable occurrence of other species (the vagrant Epitrimerus cupressi (Keifer) is common on the Mediterranean cypress in Italy), detection and monitoring of T. juniperinus require taxonomic expertise and are often time-consuming and challenging before serious damage is discernible. In the present study, a rapid, cost-effective PCR-based method was developed and validated to detect T. juniperinus on cypresses. The cytochrome c oxidase subunit I gene was amplified with degenerate and specific primers, but the latter were the only ones able to discriminate between T. juniperinus and E. cupressi. PCR products distinguished the two species both in a pool of individuals in a mixed population of both species and in single individuals, indicating the sensitivity of the detection method. PCR-RFLP (restriction fragment length polymorphism) by means of XmnI and XbaI endonucleases separated the two species. Furthermore, a washing-sieving protocol was used to make mite collection from the tree sample faster and simpler; this procedure did not interfere with the molecular detection of the species. The possibility of the routine use of this assay to monitor quarantine eriophyoids infesting plant material is discussed.

Occurrence and transmission of mycovirus Cryphonectria hypovirus 1 from dejecta of Thyreophagus corticalis (Acari, Acaridae).

The natural spread of virus-induced hypovirulence is highly involved in the recovery of blighted chestnut stands and orchards in Italy and in Europe. The potential role of corticolous mites as vectors of hypovirulence in blighted chestnut Castanea sativa (Mill.) stands was pointed out in previous reports. Here, by using RT-PCR, mycovirus Cryphonectria hypovirus (CHV1) was detected in Thyreophagus corticalis mites reared on a hypovirulent strain in monoxenic cultures and in their faecal pellets. Cryphonectria parasitica mycelium derived from mites' dejecta was able to transmit CHV1 to the virulent strain determining its conversion to hypovirulent one. This converted strain induced healing cankers on excised stems, differently from the un-converted virulent strain. Our findings prove the spread of CHV1 by corticolous mites that feed on virus-infected fungus and emphasize their potential role as vectors.

Terpene arms race in the Seiridium cardinale - Cupressus sempervirens pathosystem.

The canker-causing fungus Seiridium cardinale is the major threat to Cupressus sempervirens worldwide. We investigated the production of terpenes by canker-resistant and susceptible cypresses inoculated with S. cardinale, the effect of these terpenes on fungal growth, and the defensive biotransformation of the terpenes conducted by the fungus. All infected trees produced de novo terpenes and strongly induced terpenic responses, but the responses were stronger in the canker-resistant than the susceptible trees. In vitro tests for the inhibition of fungal growth indicated that the terpene concentrations of resistant trees were more inhibitory than those of susceptible trees. The highly induced and de novo terpenes exhibited substantial inhibition (more than a fungicide reference) and had a high concentration-dependent inhibition, whereas the most abundant terpenes had a low concentration-dependent inhibition. S. cardinale biotransformed three terpenes and was capable of detoxifying them even outside the fungal mycelium, in its immediate surrounding environment. Our results thus indicated that terpenes were key defences efficiently used by C. sempervirens, but also that S. cardinale is ready for the battle.

Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals.

The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.