Indirect plant-mediated interactions between heterospecific parasitoids that develop in different caterpillar species.

Parasitoids induce physiological changes in their herbivorous hosts that affect how plants respond to herbivory. The signature of parasitoids on induced plant responses to feeding by parasitized herbivores indirectly impacts insect communities interacting with the plant. The effect may extend to parasitoids and cause indirect interaction between parasitoids that develop inside different herbivore hosts sharing the food plant. However, this type of interactions among parasitoid larvae has received very little attention. In this study, we investigated sequential and simultaneous plant-mediated interactions among two host-parasitoid systems feeding on Brassica oleracea plants: Mamestra brassicae parasitized by Microplitis mediator and Pieris rapae parasitized by Cotesia rubecula. We measured the mortality, development time, and weight of unparasitized herbivores and performance of parasitoids that had developed inside the two herbivore species when sharing the food plant either simultaneously or sequentially. Plant induction by parasitized or unparasitized hosts had no significant effect on the performance of the two herbivore host species. In contrast, the two parasitoid species had asymmetrical indirect plant-mediated effects on each other's performance. Cotesia rubecula weight was 15% higher on plants induced by M. mediator-parasitized hosts, compared to control plants. In addition, M. mediator development time was reduced by 30% on plants induced by conspecific but not heterospecific parasitoids, compared to plants induced by its unparasitized host. Contrary to sequential feeding, parasitoids had no effect on each other's performance when feeding simultaneously. These results reveal that indirect plant-mediated interactions among parasitoid larvae could involve any parasitoid species whose hosts share a food plant.

Investigation of Trichoderma species colonization of nursery grapevines for improved management of black foot disease.

BACKGROUND: Black foot disease (BFD) is one of the main fungal diseases associated with young grapevine decline. Trichoderma holds the potential to be used as biocontrol agent against this disease, though variable success of colonization were found when applied to nursery vines in previous studies. Therefore, field experiments were established to evaluate different methods of application of Trichoderma atroviride, and to evaluate the efficacy of different commercial Trichoderma products on BFD in nursery vines post callusing over two seasons. RESULTS: Only in one season of the trial evaluating different products did all of the Trichoderma treatments significantly lower the black foot infections in the rootstock bases of the vines (mean black foot pathogen incidence of 1.00 to 2.50% in Trichoderma treated vines versus 6.50% in the untreated control). When comparing tissue parts, the base of the vine and collar roots had significantly higher Trichoderma colonization than the middle and root tip parts. Significantly less BFD pathogens were isolated from the base in comparison to the roots. These colonization trends were found for both field trials over both seasons. The different application methods showed that dipping of basal ends in the dry formulation followed by monthly soil drenches consistently gave higher colonization [mean Trichoderma incidence in the bases were 39.20% (2016/2017) and 28.00% (2017/2018)], while the 1 h soak of the bases of vines was not effective [mean Trichoderma incidence in the bases were 8.80% (2016/2017) and 4.00% (2017/2018)] and did not differ from the untreated control. CONCLUSION: Even though Trichoderma spp. were not sufficient to prevent infections by BFD pathogens, a certain degree of protection was obtained in the basal ends, which may contribute to longevity of the vines once planted in the vineyard.

A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds.

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate their incidence levels. In the present study, we designed two sets of primers that target the ß-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across ß-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. lata during the dormant season. Experimental grapevines were located in two counties of northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation method, respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata-complex in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.

Differing Alterations of Two Esca Associated Fungi, Phaeoacremonium aleophilum and Phaeomoniella chlamydospora on Transcriptomic Level, to Co-Cultured Vitis vinifera L. calli.

The filamentous fungi Phaeoacremonium aleophilum (P.al, Teleomorph: Togninia minima) and Phaeomoniella chlamydospora (P.ch) are believed to be causal agents of wood symptoms associated with the Esca associated young vine decline. The occurrence of these diseases is dramatically increasing in vineyards all over the world whereas efficient therapeutic strategies are lacking. Both fungi occupy the same ecological niche within the grapevine trunk. We found them predominantly within the xylem vessels and surrounding cell walls which raises the question whether the transcriptional response towards plant cell secreted metabolites is comparable. In order to address this question we co-inoculated grapevine callus culture cells with the respective fungi and analyzed their transcriptomes by RNA sequencing. This experimental setup appears suitable since we aimed to investigate the effects caused by the plant thereby excluding all effects caused by other microorganisms omnipresent in planta and nutrient depletion. Bioinformatics analysis of the sequencing data revealed that 837 homologous genes were found to have comparable expression pattern whereas none of which was found to be differentially expressed in both strains upon exposure to the plant cells. Despite the fact that both fungi induced the transcription of oxido- reductases, likely to cope with reactive oxygen species produced by plant cells, the transcriptomics response of both fungi compared to each other is rather different in other domains. Within the transcriptome of P.ch beside increased transcript levels for oxido- reductases, plant cell wall degrading enzymes and detoxifying enzymes were found. On the other hand in P.al the transcription of some oxido- reductases was increased whereas others appeared to be repressed. In this fungus the confrontation to plant cells results in higher transcript levels of heat shock and chaperon-like proteins as well as genes encoding proteins involved in primary metabolism.

Variations in Early Response of Grapevine Wood Depending on Wound and Inoculation Combinations with Phaeoacremonium aleophilum and Phaeomoniella chlamydospora.

Defense mechanisms in woody tissue are poorly understood, especially in vine colonized by trunk pathogens. However, several investigations suggest that molecular mechanisms in the central tissue of Vitis vinifera L. may be involved in trunk-defense reactions. In this work, the perception of Phaeoacremonium aleophilum and Phaeomoniella chlamydospora alone or together were investigated in cuttings of Cabernet Sauvignon trunks. Plant responses were analyzed at the tissue level via optical microscopy and at the cellular level via plant-gene expression. The microscopy results revealed that, 6 weeks after pathogen inoculation, newly formed vascular tissue is less developed in plants inoculated with P. chlamydospora than in plants inoculated with P. aleophilum. Co-inoculation with both pathogens resulted in an intermediate phenotype. Further analysis showed the relative expression of the following grapevine genes: PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, PIN, CAM, LOX at 10, 24, 48, and 120 h post-inoculation (hpi). The gene set was induced by wounding before inoculation with the different pathogens, except for the genes CAM and LOX. This response generated significant noise, but the expression of the grapevine genes (PAL, PR10.3, TL, TLb, Vv17.3, STS, STS8, CWinv, and PIN) still differed due to perception of mycelium by the plant. Furthermore, at 48 hpi, the induction of PAL and STS8 differs depending on the pathogen, and a specific pattern emerges from the different inductions associated with the different treatments. Based on these results, we conclude that V. vinifera L. trunk perceives the presence of pathogens differently depending on the inoculated pathogen or even on the combination of co-inoculated pathogens, suggesting a defense orchestration in the perennial organs of woody plants.

Deciphering the Niches of Colonisation of Vitis vinifera L. by the Esca-Associated Fungus Phaeoacremonium aleophilum Using a gfp Marked Strain and Cutting Systems.

INTRODUCTION: Esca disease has become a major threat for viticulture. Phaeoacremonium aleophilum is considered a pioneer of the esca complex pathosystem, but its colonisation behaviour inside plants remains poorly investigated. MATERIAL AND METHODS: In this study, P. aleophilum::gfp7 colonisation was assessed six and twelve weeks post-inoculation in two different types of tissues: in the node and the internode of one year-old rooted cuttings of Cabernet Sauvignon. These processes of colonisation were compared with the colonisation by the wild-type strain using a non-specific lectin probe Alexa Fluor 488-WGA. RESULTS: Data showed that six weeks post-inoculation of the internode, the fungus had colonised the inoculation point, the bark and xylem fibres. Bark, pith and xylem fibres were strongly colonised by the fungus twelve weeks post-inoculation and it can progress up to 8 mm from the point of inoculation using pith, bark and fibres. P. aleophilum was additionally detected in the lumen of xylem vessels in which tyloses blocked its progression. Different plant responses in specific tissues were additionally visualised. Inoculation of nodes led to restricted colonisation of P. aleophilum and this colonisation was associated with a plant response six weeks post-inoculation. The fungus was however detected in xylem vessels, bark and inside the pith twelve weeks post-inoculation. CONCLUSIONS: These results demonstrate that P. aleophilum colonisation can vary according to the type of tissues and the type of spread using pith, bark and fibres. Woody tissues can respond to the injury and to the presence of this fungus, and xylem fibres play a key role in the early colonisation of the internode by P. aleophilum before the fungus can colonise xylem vessels.