Transcriptional profiling of the terpenoid biosynthesis pathway and in vitro tests reveal putative roles of linalool and farnesal in nectarine resistance against brown rot.

The most devastating fungal disease of peaches and nectarines is brown rot, caused by Monilinia spp. Among the many plant responses against biotic stress, plant terpenoids play essential protective functions, including antioxidant activities and inhibition of pathogen growth. Herein, we aimed to characterize the expression of terpenoid biosynthetic genes in fruit tissues that presented different susceptibility to brown rot. For that, we performed artificial inoculations with Monilinia laxa at two developmental stages (immature and mature fruit) of two nectarine cultivars ('Venus' -mid-early season cultivar - and 'Albared' -late season cultivar-) and in vitro tests of the key compounds observed in the transcriptional results. All fruit were susceptible to M. laxa except for immature 'Venus' nectarines. In response to the pathogen, the mevalonic acid (MVA) pathway of the 'Venus' cultivar was highly induced in both stages rather than the methylerythritol phosphate (MEP) pathway, being the expression of some MEP-related biosynthetic genes [e.g., PROTEIN FARNESYLTRANSFERASE (PpPFT), and 3S-LINALOOL SYNTHASE (PpLIS)] different between stages. In 'Albared', both stages presented similar responses to M. laxa for both pathways. Comparisons between cultivars showed that HYDROXYMETHYLGLUTARYL-CoA REDUCTASE (PpHMGR1) expression levels were common in susceptible tissues. Within all the terpenoid biosynthetic pathway, linalool- and farnesal-related pathways stood out for being upregulated only in resistant tissues, which suggest their role in mediating the resistance to M. laxa. The in vitro antifungal activity of linalool and farnesol (precursor of farnesal) revealed fungicidal and fungistatic activities against M. laxa, respectively, depending on the concentration tested. Understanding the different responses between resistant and susceptible tissues could be further considered for breeding or developing new strategies to control brown rot in stone fruit.

Light Intensity Alters the Behavior of Monilinia spp. in vitro and the Disease Development on Stone Fruit-Pathogen Interaction.

The development of brown rot caused by the necrotrophic fungi Monilinia spp. in stone fruit under field and postharvest conditions depends, among others, on environmental factors. The effect of temperature and humidity are well studied but there is little information on the role of light in disease development. Herein, we studied the effect of two lighting treatments and a control condition (darkness) on: (i) several growth parameters of two Monilinia spp. (M. laxa and M. fructicola) grown in vitro and (ii) the light effect in their capacity to rot the fruit (nectarines) when exposed to the different lighting treatments. We also assessed the effect of such abiotic factors in the development of the disease on inoculated nectarines during postharvest storage. Evaluations also included testing the effect of fruit bagging on disease development as well as on ethylene production. Under in vitro conditions, lighting treatments altered colony morphology and conidiation of M. laxa but this effect was less acute in M. fructicola. Such light-induced changes under in vitro development also altered the capacity of M. laxa and M. fructicola to infect nectarines, with M. laxa becoming less virulent. The performance of Monilinia spp. exposed to treatments was also determined in vivo by inoculating four bagged or unbagged nectarine cultivars, indicating an impaired disease progression. Incidence and lesion diameter of fruit exposed to the different lighting treatments during postharvest showed that the effect of the light was intrinsic to the nectarine cultivar but also Monilinia spp. dependent. While lighting treatments reduced M. laxa incidence, they enhanced M. fructicola development. Preharvest conditions such as fruit bagging also impaired the ethylene production of inoculated fruit, which was mainly altered by M. laxa and M. fructicola, while the bag and light effects were meaningless. Thus, we provide several indications of how lighting treatments significantly alter Monilinia spp. behavior both in vitro and during the interaction with stone fruit. This study highlights the importance of modulating the lighting environment as a potential strategy to minimize brown rot development on stone fruit and to extent the shelf-life period of fruit in postharvest, market, and consumer's house.

Depicting the battle between nectarine and Monilinia laxa: the fruit developmental stage dictates the effectiveness of the host defenses and the pathogen's infection strategies.

Infections by the fungus Monilinia laxa, the main cause of brown rot in Europe, result in considerable losses of stone fruit. Herein, we present a comprehensive transcriptomic approach to unravel strategies deployed by nectarine fruit and M. laxa during their interaction. We used M. laxa-inoculated immature and mature fruit, which was resistant and susceptible to brown rot, respectively, to perform a dual RNA-Seq analysis. In immature fruit, host responses, pathogen biomass, and pathogen transcriptional activity peaked at 14-24 h post inoculation (hpi), at which point M. laxa appeared to switch its transcriptional response to either quiescence or death. Mature fruit experienced an exponential increase in host and pathogen activity beginning at 6 hpi. Functional analyses in both host and pathogen highlighted differences in stage-dependent strategies. For example, in immature fruit, M. laxa unsuccessfully employed carbohydrate-active enzymes (CAZymes) for penetration, which the fruit was able to combat with tightly regulated hormone responses and an oxidative burst that challenged the pathogen's survival at later time points. In contrast, in mature fruit, M. laxa was more dependent on proteolytic effectors than CAZymes, and was able to invest in filamentous growth early during the interaction. Hormone analyses of mature fruit infected with M. laxa indicated that, while jasmonic acid activity was likely useful for defense, high ethylene activity may have promoted susceptibility through the induction of ripening processes. Lastly, we identified M. laxa genes that were highly induced in both quiescent and active infections and may serve as targets for control of brown rot.

Dispersion, persistence, and stability of the biocontrol agent Penicillium frequentans strain 909 after stone fruit tree applications.

The capacity of dispersion, persistence, and stability from biocontrol agents is essential before these organisms can be developed into a commercial product. Interactions that microorganisms establish with stone fruit trees may be beneficial in the exploitation of trees in agriculture as crop production. The natural background levels of Penicillium frequentans strain 909 dispersion, persistence, and stability were assessed after tree applications and postharvest conditions. A fingerprinting-based approach to trace genetic stability of P. frequentans along stored time and its release in the field was developed. P. frequentans was successfully traced and discriminated. This strain was dispersed well in treated trees, persisting in the ecosystem up to 2 weeks and staying genetically stable after 36 months of storage. However, the dispersal of P. frequentans was very limited on around untreated trees and soil. P. frequentans dispersed randomly into the air, and its presence reduced from the first day to disappear completely at 15-21 days after application. Great losses of P. frequentans and its increased dispersal in open field conditions probably resulted from rainfall. Biological control strategies with Pf909 were discussed.

Environmental fate and behaviour of the biocontrol agent Bacillus amyloliquefaciens CPA-8 after preharvest application to stone fruit.

BACKGROUND: Bacillus amyloliquefaciens strain CPA-8 has been described as an effective biocontrol agent to control brown rot in stone fruit for both preharvest and postharvest applications. However, no information about the environmental fate and behaviour of this strain under field conditions is available. RESULTS: The dispersion of the CPA-8 application was evaluated using water-sensitive papers, and complete coverage was observed on the leaves of treated trees, while <1% of non-treated tree leaves had CPA-8. CPA-8 persisted on the fruit of treated trees during preharvest and postharvest conditions, while a significant decrease on leaves and weeds was observed 21 days after treatment. On non-treated trees, CPA-8 was detected on leaves until 180 days after treatment, and on weeds, the CPA-8 population was dependent on the distance from the treated trees. A high persistence of CPA-8 was detected on inert materials, such as clothes and gloves worn by handlers and plastic harvesting boxes. More than 99% of the samples with a CPA-8 phenotype were confirmed as CPA-8 using polymerase chain reaction (PCR). CONCLUSION: This work demonstrated a good distribution, persistence and adaptation of the CPA-8 strain to field and postharvest conditions. Monitoring of dispersion and persistence is an excellent tool to determine the time of application and provides valuable information for registering issues. © 2017 Society of Chemical Industry.