Fumigant concentration and weed density data were subjected to nonlinear regression analysis using Sigma Plot v. 11 .Because application rates tested in 2007 were normal, the rates were sufficiently high to suppress most pathogens and weeds regardless of the film permeability. For this reason, in 2008 we chose to compare fumigant retention under the two films at a range of rates from low to high, to determine if TIF would improve retention and efficacy across that range. At Salinas in 2008, 1,3-D plus chloropicrin concentrations in the 200-poundper-acre treatment were higher 24 hours post-application under TIF than under standard film . The 1,3-D plus chloropicrin concentrations in the 300-pound-per-acre treatment were higher under TIF than under standard film at 8, 24, 48 and 96 hours after application.Generally, there were no tarp effects on plant diameters except at the 1,3-D plus chloropicrin rate of 100 pounds per acre; TIF plants were 9.4 inches compared with 8.3 inches for standard-film plants .The differences were significant in the 1,3-D plus chloropicrin treatments at 100 and 200 pounds per acre . There was a positive correlation between the 8-hour 1,3-D plus chloropicrin concentration and full season fruit yields for each film . The 8-hour fumigant concentration accounted for 49% to 55% of yield variability in the standard and TIF treatments, respectively. Weed densities were higher under standard film than under TIF. At 100 pounds per acre, 1,3-D plus chloropicrin applied under TIF had significantly fewer weeds than the same rate under standard film . The interaction between fumigant rate and film was significant for common chickweed and common purslane ,dutch bucket hydroponic meaning that the survival of each of these two weeds was different under the two films.
The interaction of yellow nuts edge rate by film was not significant , indicating that nuts edge survival was similar under both films. However, we sought to describe the performance of TIF, therefore we evaluated nuts edge separately under both films. Yellow nuts edge tuber survival was less under TIF than standard film at 100 pounds per acre 1,3-D plus chloropicrin, but not at the other rates. Common purslane and common chickweed seed survival were lower under TIF than standard film at 50 pounds per acre 1,3-D plus chloropicrin . Little mallow and knot weed viability were similar under both films . Differences in weed control due to film type were only observed at the lower fumigant doses of 50 and 100 pounds per acre. This is likely due to the fact that TIF retained more fumigant than standard film, which resulted in a higher dose and lower weed seed survival under TIF than standard film . At application rates above 100 pounds per acre, the fumigant concentrations under both TIF and standard films were sufficiently high to kill weeds, so no differences were found between the films.Results of two trials conducted over 2 years indicate that TIF consistently held methyl bromide plus chloropicrin and 1,3-D plus chloropicrin at higher concentrations than standard film . At fumigant rates of 100 and 200 pounds per acre, strawberry fruit yields were higher and weed control was more complete where TIF was used, compared to standard film . This is likely due to the higher fumigant concentrations being held for a longer time under the TIF than under the morepermeable standard film, so that weeds and possibly soil pathogens were more thoroughly controlled. Drip-applied 1,3-D plus chloropicrin under standard film required at least 300 pounds per acre to provide fruit yields comparable to methyl bromide plus chloropicrin . In contrast, 1,3-D pluschloropicrin drip-applied under TIF at 200 pounds per acre had fruit yields and weed control similar to methyl bromide plus chloropicrin, a 33% reduction in 1,3-D plus chloropicrin rate compared to standard film. Similarly, Ajwa et al. found that the rates of drip-applied chloropicrin required to produce strawberry yields similar to methyl bromide plus chloropicrin were 294 and 198 pounds per acre under standard and VIF, respectively, a 48% reduction in chloropicrin. The recent registration of methyl iodide as a soil fumigant by the California Department of Pesticide Registration requires the use of impermeable films . Methyl iodide must be used with impermeable films as approved by CDPR, and TIF is on the list of approved films .
The results presented here further validate that TIF is effective at increasing fumigant retention and may ease some of the burdens of fumigant regulations on end-users, as well as ease concerns of the general public about exposure to fumigants.Monilinia laxa is the main causal agent of brown rot in Europe, leading to important losses of stone fruit in the field and post harvest. The worldwide yearly losses are estimated to be 1.7 M euros for peach and nectarine and 170 M USD for peach, cherry, and plum production. The disease is controlled using several cultural practices , chemical fungicides in the orchard, treatments onto mummified fruit, and post harvest storage at low temperatures. However, the gradual withdrawal of some fungicides driven by concerns about their negative impact on the environment and human health, the constant threat of the emergence of fungicide resistance, and the appearance of novel virulence alleles demonstrate the need for alternative methods for managing brown rot. Prior to infection, M. laxa can remain latent or quiescent on flowers and fruit surfaces until favorable host factors , and environmental factors and other characteristics intrinsic to the stone fruit variety, trigger the disease cycle. During fruit infection, M. laxa can overcome the need for wounds to infect and penetrate the plant cell. As a necrotrophic pathogen, M. laxa relies on the secretion of cell wall-degrading enzymes , such as pectin methyl esterases, and possibly phytotoxins, although these compounds have not been fully identified yet. After penetration, M. laxa colonizes the epidermis of the fruit with hyphae causing the collapse and disruption of cells, lysogenic cavities, and total degradation of the cuticle and epidermis, similar to the lesions caused by M. fructicola. Overall, fruit can be infected at any growth stage, but their susceptibility to brown rot increases with maturation, which results in a short post harvest life. Hence, the activation of immune responses alongside the physicochemical properties of the fruit may determine the pathogen’s ability to infect and spread.
Although these underlying mechanisms have not been fully elucidated, possible explanations could depend on changes in cell wall composition, volatiles, organic acids, and phenolic compounds. We hypothesize that M. laxa is able to adapt its infection strategies according to the nectarine developmental stage, resulting in either quiescent or disease progression, while the plant host can only establish effective defenses to restrict pathogen growth in fruit tissues that have not yet reached full maturity. Here, the fruit responses and pathogenicity mechanisms in the nectarine–M. laxa interaction were investigated as a function of the host developmental stage and time. Nectarine fruit was harvested at two different developmental stages and inoculated with M. laxa. Disease development and ethylene production were assessed for 3 days. Thanks to the recent availability of the M. laxa 8L genome, a comparative transcriptomics study was conducted on the nectarine–M. laxa pathosystem across four time points. This approach allowed us to identify not only host defense responses that were uniquely or highly induced in immature fruit during early infections,dutch buckets system which may partially explain why these tissues are resistant to brown rot, but also key strategies employed by the fungus to either become established in tissues or colonize them, which may be targeted to control brown rot.We visually assessed the development of brown rot over time at two maturity stages of nectarine . Quality parameters were measured and summarized in Supplementary Table S1. Overall, the disease progressed in mature tissues, while only surface discoloration was observed in immature tissues. At the mature stage, tissue maceration was observed on the surface of the fruit at 14 hpi followed by the pathogen penetration of the pericarp tissues between 14 and 24 hpi, and increasing lesion spread at 48 and 72 hpi. Fungal biomass was also estimated in both inoculated and control fruit to complement the visual assessments . Although no symptoms of brown rot disease were visible on the immature fruit surface at any time point, the M. laxa biomass increased from 6 to 14 hpi, when the highest quantity was detected, and then significantly decreased until 72 hpi. Although at early stages of infection , the fungal biomass was not significantly different between immature and mature tissues, it increased exponentially in the mature fruit at later time points, reaching levels approximately twenty times more than the maximum observed in immature fruit. In control tissues, a negligent quantity of the fungal biomass was detected across all time points in both stages. A dual RNA-Seq study revealed the dynamics of the fruit–pathogen interaction at early and late infection time points. The expression of 21,334 nectarine genes and 8364 M. laxa genes was detected across all developmental stages and time points .
The proportion of the total mapped reads for each sample that corresponded to M. laxa strongly correlated with the measurements of fungal biomass. Remarkably, more than 6000 genes were found to be expressed in inoculated immature fruit at 14 hpi and 24 hpi, indicating that the pathogen was active in these tissues but yet it could not cause disease. More genes were detected in mature fruit, increasing across time, from 6565 at 6 hpi up to 8287 at 48 hpi, reflecting the progression of pathogen growth and host tissue colonization.The principal component analyses revealed that in nectarine, PC1 and PC2 clearly separated the samples based on their developmental stage and infection status . Notably, at both development stages, 14 hpi was the time point when the inoculated samples appeared to experience a significant change in their expression profiles compared to the controls. These results demonstrate that early time points are critical for dictating the outcome of the interaction. For M. laxa, PC1 distinguished the samples based on the fruit developmental stage, while PC2 mainly divided the samples between early- and lateinoculation time points . In immature fruit, there was an evident switch in the pathogen’s transcriptional profile after 14 hpi, coinciding with the decrease in fungal biomass, and then continued to change up to 48 hpi. In mature fruit, M. laxa showed a change in gene expression between 6 and 14 hpi, when disease symptoms were first noticed on the fruit surface. Then, between 14 and 24 hpi, the pathogen altered its gene expression in mature fruit once again and retained most of these changes up to 48 hpi. Remarkably, the expression patterns of M. laxa at late time points of infection were highly divergent when infecting immature and mature tissues, suggesting that the pathogen utilizes different survival or infection mechanisms depending on the host developmental stage. A differential gene expression analysis was performed to determine the responses of immature and mature fruit to M. laxa, and to identify specific strategies used by the pathogen at specific times of infection. Nectarine DE genes were identified in comparisons between inoculated and control fruit for each maturity stage and time point . A total of 4005 DEGs were detected in immature fruit across all time points, and of these the majority were upregulated in inoculated tissues. In immature fruit, the number of DEGs progressively increased over time and peaked at 24 hpi; then, the changes in gene expression appeared to reach a slightly lower plateau at 48 hpi. Mature fruit displayed a stronger transcriptional response to M. laxa infection since a total of 13,855 DEGs were detected at early and late time points. The number of DEGs in mature fruit continuously increased from 6 hpi to 48 hpi, indicating that the host tissues were undergoing a large transcriptional reprograming as the disease progressed. Monilinia laxa DEGs were detected by comparing the expression profiles of the fungus at each time point against 6 hpi for immature and mature fruit, respectively . These comparisons allowed us to depict how the pathogen modified its transcriptional response based on the initial time point of the interaction when gene expression profiles of M. laxa were similar between immature and mature fruit .To study host metabolic pathways altered during M. laxa progression, we performed a functional enrichment analysis for KEGG terms in the upregulated nectarine DEGs at each time point for immature and mature fruit . Figure 3a depicts KEGG terms that were significantly enriched in at least four out of the eight comparisons .