Especially prominent in F. acuminatum and R. stolonifer were proteins with similarity to subtilisin-like proteases. This family of enzymes is mostly associated with plants and particularly plant defense, but subtilisin-like proteases involved in pathogenicity have been described for fungi as well . Fungal plant pathogens are also known to express inhibitors of these types of proteases as a counterdefense . Since these inhibitors possess sequence similarity to the proteases themselves, the enzymes identified in F. acuminatum and R. stolonifer may be inhibitors, proteases, or a mixture of both. Additionally, proteases can help with host tissue decomposition by breaking down cell wall structural proteins or can serve in degradation of proteins to provide a source of nutrition for fungal growth . For example, the saprotrophic fungal species Verticillium albo-atrum and V. dahliae were described to secrete proteases to break down structural proteins that stabilize the plant cell walls . High proteolytic activity resulting in the degradation of proteins into free amino acids was also reported during fermentation of tempeh by several Rhizopus species . Botrytis cinerea, F. acuminatum, and R. stolonifer also make use of a variety of CAZymes during interactions with the host. Several CAZyme families are involved in the breakdown of physical barriers present in the host tissues, greenhouse vertical farming namely the various cell wall components , cell wall reinforcements , and the waxy fruit cuticle.
Many of these enzymes, such as polygalacturonases, pectin methylesterases, pectate lyases, and endo-β-1,4-glucanases, mirror the activities of host enzymes active during the ripening-related softening of the fruit . Others, such as cellulases, cutinases, and lipases, degrade components that are not typically degraded during ripening. Production of cellulases is also coupled with enzymes involved in degradation of cellobiose, the disaccharide product of cellulose breakdown. Both B. cinerea and F. acuminatum appear to focus on production of these latter CAZyme families in MG fruit more than in RR fruit. This may be due to the greater strength and integrity of the cell wall in MG fruit, which requires the fungus to mount a larger attack on the physical barriers in order to penetrate into the cells. Degradation of pectin is a hallmark feature of B. cinerea infection of plant tissues . The principal enzymes responsible for this process are polygalacturonases , pectin methylesterases , and pectate lyases . Both PGs and PLs cleave the α- 1,4-linkages in the homogalacturonan backbone of pectins. PMEs catalyze the removal of methylester groups on the C6 carbons of galacturonan, which allows for further degradation by PGs. Although overexpression of PME inhibitors in Arabidopsis leaves has been shown to increase resistance to B. cinerea , mutations in Bcpme1 and Bcpme2 do not appear to affect virulence in tomato leaves . In B. cinerea, all three classes of enzymes appear to be highly expressed in MG fruit but not as prominently in RR fruit.
Not only do the GH28, PL1-7, and PL3-2 families constitute a greater fraction of upregulated CAZymes in MG fruit, but for PGs, PLs, and PMEs that are commonly upregulated in MG and RR fruit, upregulation is consistently greater in MG fruit over RR fruit. Additionally, although no F. acuminatum PGs were detected in MG, the two upregulated PMEs, FacuDN5818c0g1i1 and FacuDN10179c0g1i1, were only active in MG fruit. Moreover, PL1-7 and PL3-2 genes were strongly expressed in MG fruit, with one PL3- 2 gene, FacuDN8473c0g1i1, showing a log2FC of 10.29 at 1 dpi, the highest of any plant CWDE in this treatment. Only one R. stolonifer PG, RstoDN2036c0g1i1, was detected in MG fruit. However, given that this single R. stolonifer PG was one of only two CAZymes found in 1 dpi MG fruit, it is reasonable to believe PG activity in R. stolonifer isbeing underestimated due to low sequence coverage of fungal transcripts in this treatment. The absence of upregulation of any R. stolonifer pectate lyases in any fruit further underscores this point. Given the prominence of pectin degradation in B. cinerea and F. acuminatum, a more targeted analysis of R. stolonifer pectin degradation, especially in MG fruit, is warranted. Degradation of the host cell wall in MG fruit by pathogen enzymes may accelerate ripening and in turn facilitate a more favorable environment for colonization. Pectinderived oligosaccharides have been shown to induce ethylene production in tomato fruit , which further upregulates expression of host CWDEs, including PG. B. cinerea can synthesize its own ethylene via the α-keto-γ-methylthiobutyric acid pathway , though it is still unknown whether the pathogen produces ethylene during interactions with the fruit. Ethylene production during plant infection has also been reported via the KMBA pathway for species of Fusarium , but not, to our knowledge, for R. stolonifer. However, the specific genes involved in the KMBA pathway in B. cinerea or Fusarium spp. have yet to be elucidated.
As colonization proceeds, sugar substrates become available due to degradation of cell wall polysaccharides as well as increased access to stored sugars in the fruit. As a consequence, fungi actively infecting RR tomato fruit induced enzymes that metabolize simple sugars. Sugar metabolism is accompanied by expression of CAZyme families involved in the production and modification of chitin, the structural component of fungal cell walls. Chitin production is known to be a hallmark of growth for fungal pathogens . Interestingly, chitin production and modification appear to be prominent not only in RR fruit for each pathogen, but also in MG fruit inoculated with F. acuminatum, where a much greater amount of mycelia growth was observed compared to the other two pathogens. The equal representation of CE4 enzymes in MG and RR fruit inoculated with F. acuminatum is reflective of the ability of this fungus of producing hyphae at either fruit ripening stage. The abundance of polysaccharide-building glycosyltransferases in RR infections with R. stolonifer is also likely connected to the abundant mycelial growth. Other CAZyme families represent more specialized roles in the infection process. Production of enzymes in the AA7 family may be related to the production of polyketide toxins in B. cinerea and R. stolonifer. B. cinerea is known to produce botcinic acid, a polyketide mycotoxin, during infection . However, the AA7 genes detected to be upregulated in fruit infection here are not known members of the botcinic acid pathway, suggesting that B. cinerea may produce additional uncharacterized polyketide mycotoxins during fruit infection. Even though upregulated F. acuminatum genes involved in toxin production are not annotated as members of the AA7 family, fumonisins are products of polyketide metabolism . The observed upregulation of fumonisin biosynthesis related genes indicates that F. acuminatum also produces polyketide mycotoxins during infection of unripe and ripe tomato fruit. However, we also observed upregulation of biosynthetic genes involved in production of trichothecenes , nft vertical farming which indicates that F. acuminatum also relies on other toxins during infection of tomato fruit concordant with the classification of F. acuminatum as strong toxin producer . Additionally, the AA6 family that appears during RR infections of F. acuminatum and R. stolonifer may be involved in metabolism of host defense compounds. These enzymes are 1,4-benzoquinone reductases, which have been shown to function in fungal protection against destructive host-produced quinones . Another physiological factor which may influence the success of infection is the pH of the pathogen-host interface. As the tomato fruit ripens, the apoplast becomes more acidic . Furthermore, B. cinerea has been shown to acidify the host environment through the production and secretion of oxalic acid . A key enzyme in oxalic acid biosynthesis is BcOAH1 , which encodes oxaloacetate hydrolase . However, there is significant down regulation of this gene in RR fruit compared to MG fruit. This suggests that, if B. cinerea utilizes oxalic acid to acidify tomato fruit, it does so to a much lesser extent in RR fruit where the pH is already comparatively acidic. In contrast, during infection of Arabidopsis roots, F. oxysporum relies on alkalinization via peptides known as rapid alkalinizing factors . However, a BLAST search of RALF sequences, as was performed to identify fungal RALFs in Thynne et al. , revealed no clear RALF genes in our transcriptome of F. acuminatum.
The importance of fruit ripening for the success of fungal infections was confirmed by comparing fungal growth and disease development in fruit from wild-type and a non-ripening mutant after fungal inoculation. Growth and morphology of B. cinerea, F. acuminatum and R. stolonifer on nor MG and RR-like tomato fruit was comparable to that on wild-type MG fruit. This result is in agreement with our previous report that nor tomato fruit is resistant to B. cinerea infections . The inability to infect non-ripening tomato fruit highlights the dependency of these fungi on the activation and progression of ripening events that transform the host tissues into a favorable environment for disease development. Altogether, our results confirm that infection success of the three pathogens B. cinerea, F. acuminatum and R. stolonifer largely depends on fruit ripening stage. This is due to all three pathogens sharing similar lifestyles and necrotrophic infection strategies. However, the capacity to infect different plant tissues differs between the three fungi. B. cinerea shows distinct strategies in both ripening stages likely due to its ability to induce susceptibility in the host , whereas R. stolonifer is active almost exclusively in RR fruit. The ability of F. acuminatum to infect both MG and RR fruit may be reflective of its especially wide host range, which includes insects in addition to fruit . A summary of infection strategies utilized by the three pathogens during infection of MG and RR tomato fruit is shown in Table 2. Further research on which processes identified are required for successful infection would lead to a greater understanding of fruit-pathogen interactions and, ultimately, strategies for their management.In the simplest possible terms, Alternate Bearing is a two year cycle of fruit production that occurs in perennial plants, and is best known from fruit-bearing trees like apples and avocados. In one year, the plants will produce many fruits, while in the second year, they produce very few. This biennial pattern then often repeats itself in the following years creating a repetitive cycle, revealing itself as a saw-toothed line when fruit yields are plotted over many years . The fluctuation in yield is usually quite moderate and only rarely reaches the extreme values of 0% and 100%, but even minor variations can readily be detected in commercially grown orchard trees. The phenomenon is not limited to trees though, as similar biennial cycles have also been documented to occur in perennial herbs, monocots, and forest trees, which suggest that alternate bearing is a fairly common, if seldom seen plant behavior. From an economic standpoint though, the presence of alternate bearing in commercial orchards is considered to be undesirable for many reason. Not only do alternating trees tend to produce less fruit on average than regular-bearing varieties, the resulting fruit often display characteristic variation in size and appearance that reduces their market value. For example, large crops tend to produce small and poor quality fruits, while small crops can occasionally produce large and abnormally swollen fruit. Many fruits are sorted into sizes according to their intended market, where this variation makes it more difficult for the farmer to produce sufficient numbers of each. Harvest costs are largely the same whether the crop is large or small, and large crops often incur extra processing and storage fees when they can’t be sold immediately. In addition, alternate bearing trees are prone to synchronization, during which all of the trees in the orchard fluctuate in lock-step with each other. Frequently attributed to a late flower-destroying frost or other temperature anomalies, such synchronization can occur over a wide range of scales, ranging from isolated trees, to whole orchards, and even entire geographic areas . Fruit production in synchronized areas tends to saturate the market with low-quality fruit in one year, and provide little or no fruit in the second, thus limiting the potential profit in both years of the cycle. Although there are horticultural techniques available to force the trees to become regular bearing again, these introduce additional expenses in the form of time, labor, and materials. Thus the combined effects of yield, quality, irregular production, and additional expenses can easily make alternate bearing crops uneconomical to produce, despite any other attractive qualities they might have.