Jasmonic acid and ET are known to be involved in defense responses against necrotrophs, such as mediating the host’s responses against them, but ET is also required for fruit ripening and senescence processes, which are conducive to disease susceptibility. Jasmonic acid can also mediate the disease resistance of fruit by increasing the fruit antioxidant capacity, but some fungi are able to hijack the JA signaling pathway to cause disease. Although the early steps of JA biosynthesis were highly induced upon M. laxa inoculation, downregulation of receptor genes was observed in mature fruit inoculated with M. laxa when compared to controls. These findings suggest that M. laxa could be somehow blocking the JA signaling pathway, although the mechanisms involved are unknown. Ethylene biosynthesis increases during ripening of climacteric fruit, such as nectarines. In our study, the control immature fruit produced basal ethylene levels, whereas ethylene production in control mature fruit increased through time after harvest. In inoculated immature fruit, there was a significant peak of ET production as compared to the control at 24 hpi. This discrete induction of ET can be part of the fruit defense responses against M. laxa.
Alternatively,dutch bucket for tomatoes the pathogen could be inducing fruit ethylene biosynthesis in immature fruit to accelerate ripening, in an attempt to promote fruit physicochemical changes that are conducive to disease. Along this line, ACS2 and ACO1, involved in system 2 ET production, were over expressed in inoculated immature tissues. Previous studies have reported on a similar modulation of ET biosynthesis by the pathogen. However, after 24 hpi, ethylene levels in inoculated immature fruit fell to control levels, and the fruit remained resistant. This may be in part due to the upregulation of the ethylene signaling inhibitors EBF1/2, which could mitigate the ethylene-induced ripening processes that contribute to susceptibility. In contrast, in inoculated mature fruit, ET production and signal transduction were lower at 6 hpi in inoculated fruit but grater from 24 hpi onward, following the autocatalytic system 2 ethylene biosynthesis. Overall, the results indicate the ability of M. laxa to differentially alter ET production to promote susceptibility and, in turn, the ability for immature fruit, but not mature fruit, to mitigate the consequences of this induction. The above observations indicate that although M. laxa was deploying some strategies to infect the immature tissues, it was not able to overcome either the surface or the active defense responses deployed by the immature fruit. Monilinia laxa remained on the immature tissue, increasing its biomass and multiplying on the surface, until 14 hpi when it ceased to grow.
It is known that Monilinia spp. can remain quiescent on fruit surfaces and that they can employ appressoria as resting structures on immature nectarines. After 14 hpi, M. laxa biomass and reads started to decrease, switching its transcriptional machinery by employing different sets of genes in order to deploy different strategies to survive on the fruit’s surface. Some results point out that M. laxa could either be starting a quiescence period or moving toward an autolysis process, breaking cells to feed on its remains. Another possibility is that the remaining M. laxa cells on immature fruit were being attacked by the host defenses. This is supported by the expression of M. laxa genes associated with response to oxidative stress at late time points, such as catalases, previously reported in detoxification during infection of tomato leaves by B. cinerea. Thus, it is likely that immature fruit was generating reactive oxygen species during the interaction through an oxidative burst to kill the pathogen. Monilinia laxa could also be producing ROS for its development and as a pathogenicity mechanism to damage the host tissue. Particularly, the NADPH oxidase complex is involved in both fungal ROS production and its use in sclerotia development and virulence. Some genes encoding the Nox regulator R were found to be upregulated at 24 hpi in both mature and immature tissue. At later stages, a highly induced alcohol oxidase expressed in immature tissue at 48 hpi could be another ROS producer, previously described as an alternative ROS production system. Lin et al. demonstrated that AOX1 was involved in pathogenicity and oxygen stress responses in B. cinerea.
Concomitantly, nectarine counteracted the pathogen oxidative burst by expressing genes of antioxidant metabolism compounds such as glutathione and redox-related amino acids . Plant secondary metabolites such as terpenoids have been described to protect the fruit under biotic and abiotic stresses, although their role can be tissue dependent. Overall, the enrichment of genes involved in secondary metabolite biosynthesis was higher in resistant immature than susceptible mature tissue, which suggests that either the host was producing terpenoids in the resistant immature tissue to prevent the attack or that M. laxa was inhibiting its biosynthesis on mature tissue. Monilinia laxa could also be able to degrade and transform terpenoids as described for B. cinerea. The phenylpropanoid metabolism is also triggered in response to brown rot. In both immature and mature fruit, from 14 hpi to 48 hpi, phenylpropanoid-related pathways were highly induced. While on the immature tissue, these pathways could be involved in reinforcing the cell wall through lignin production, the role in the mature fruit could be more focused on the detoxification of fungal ROS production. Nevertheless, these hypotheses need to be further tested. On mature nectarines, M. laxa deployed other virulence factors in addition to ROS production and scavenging. The pathogen expressed upregulated DEGs related to proteolytic activity, containing domains such as the Pro −kumamolisin domain . The list of genes summarized in Table 1 could be putative pathogen target genes as they were expressed only when M. laxa infected the mature tissues, as none of the top five upregulated genes in mature tissue was found in the immature fruit. For instance, the highest expressed protease at in all time points is a homolog of a nonaspartyl protease found during pathogenesis in Sclerotinia sclerotiorum. Cell wall-degrading enzymes are commonly produced by necrotrophic fungi as virulence factors and their secretion by Monilinia spp. on culture media has been previously reported. A rhamnogalacturonan hydrolase ,blueberry grow pot which was highly expressed at both 24 and 48 hpi, was already described as a putative virulence factor in M. laxa infecting peaches. Current information regarding the strategies utilized by either Monilinia spp. or stone fruit or during their interaction is mainly focused on specific metabolic pathways or actions developed by one of the two players. As a novel feature of the present research, we demonstrated the synchronized responses from nectarine and M. laxa, by utilizing a resistant immature and susceptible mature fruit throughout a course of infection. Future research studies should be focused on delving into the host defense system for the ongoing development of nectarine cultivars with increased resistance to brown rot, as well as conducting in-depth fungal studies to alter the ability of M. laxa to cause disease.“Venus” nectarines Schneider were obtained from an organic orchard located in Raïmat . Fruit was bagged 6 weeks before the last harvest and then harvested at two different fruit developmental stages, “mature” and “immature” , and used immediately after harvest.
Injured or deformed fruit was discarded, and fruit for analysis was further homogenized by using a portable DA-Meter , based on the single index of absorbance difference . Other assessments of quality parameters were performed on 20 randomly selected fruit , according to the method of Baró-Montel et al..The M. laxa single-spore strain 8L was used for all experiments. Fungal conidial suspensions were maintained and prepared, as described by Baró-Montel et al..Each fruit was inoculated with the application of six 30- μL drops of a conidial suspension at a concentration of 106 conidia mL−1 on the fruit surface. Mock-inoculated fruits were equally treated with sterile water containing 0.01% Tween-80. Fruit were placed in closed containers with a relative humidity of 97 ± 3% at 20 ± 1 °C.Six cylinders of peel and pulp tissue encompassing the inoculation sites were sampled from each fruit and pooled for each replicate. Samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C until extraction. For symptom analysis, inoculated fruit was imaged at the set time points. Ethylene production of both mock and M. laxa inoculated immature and mature fruit was determined, as described by Baró-Montel.In the high input year round strawberry and vegetable cropping systems of coastal California, organic growers have to balance their desire to reduce environmental impacts with the multiple challenges of maintaining economically sustainable yields. These challenges include providing adequate plant nutrients while minimizing losses to protect water quality, maintaining low levels of disease, and building effective system-based pest management strategies. In comparison with conventionally managed systems, little research on organic systems has been available for growers to help meet these challenges and so in 2004 we formed a research-extension-grower network to develop and implement a collaborative organic research program . The California Collaborative Research and Extension network which we report on here grew out of this initial program. The first 4-5 years of research, known as the Organic Research Network Project, was funded by the USDA-Organic Agriculture Research and Extension Initiative program in 2004 , with the goals of building an organic research and extension network to support organic vegetable and strawberry producers in the region and to develop integrated fertility and pest management strategies to minimize negative impacts of agriculture on surrounding natural ecosystems and improve the economic viability of organic farming. Multiple field experiments were carried out including cover crop/fertility trials , a strawberry rotation trial , studies of the role of trap crops and hedgerows in arthropod biological control , and development of anaerobic soil disinfestation as a technique for controlling soil borne diseases in strawberry production . The current CAL-CORE program represents an extension and expansion of this earlier effort, bringing additional researchers, farmers, regions, and organizations into the network. Our goal is to provide a dynamic platform for network members to collaboratively research and evaluate integrated systems approaches to improve environmental sustainability and economic viability in a comprehensive manner. Using the information generated previously, a series of field experiments have been undertaken to examine the effect of crop rotations and specific management practices on productivity and economics as well as ecosystems services such as nutrient cycling; pest, disease, and weed suppression; soil carbon sequestration; and greenhouse gas emissions. A combination of direct measurement, modeling and Life Cycle Analysis are being used to describe the environmental imprint of the management systems being tested. The centerpiece of the project is a replicated rotation study being conducted at the University of California, Santa Cruz, Center for Agroecology and Sustainable Food Systems farm site which has been under organic management for 40 years. In addition a subset of the treatments in this study were chosen by six farmer collaborators to test on their fields. A number of additional satellite studies are also underway, but here we report on some of the preliminary findings from the main rotation study which is now in its 4th year and reflect on the functioning of the network as a mechanism for collaborative research and outreach. The goals of this study are to compare rotation length and crop composition on soil fertility and soil borne disease dynamics when used alone or in conjunction with soil amendments or anaerobic soil disinfestation. The CASFS field is an Elkhorn sandy loam , and the experiment is a split-split plot design with four replicates. It compares two and four year vegetable/strawberry rotations using combinations of crops believed to be either suppressive of a major soil borne disease , or more profitable but more conducive to disease. Superimposed on the rotations are fertility treatments : legume/cereal cover crop only, legume/cereal cover crop + compost + additional fertility amendments, cereal cover crop + mustard seed meal, or untreated control. In the two legume/cereal cover cropped treatments anaerobic soil disinfestation is used for disease management prior to planting strawberries. The study is in year 4 and all treatments were planted to strawberries in 2014. Winter cover crops in treatments 1a, 2a, 5a, 6a, 1b, 2b, 5b, and 6b were a mixture of bell bean 45%, purple vetch 45%, and cereal rye 10% and were planted at a rate of 367 kg ha-1 in November each fall and incorporated with a spader in April to May. In treatments 2a, 6a, 2b and 6b, compost and organic fertilizer were added pre-plant for each crop.