ABA, IAA and ethylene accumulation are altered by polyamine levels, which are positively correlated with fruit susceptibility to B. cinerea during strawberry ripening . Other hormones, such as brassinosteroids and jasmonic acid are present at lower levels during strawberry ripening. BR positively regulates vitamin C levels, sugar and anthocyanin biosynthesis during ripening, while negatively regulating acidity and concentration of other phenolic compounds .Endogenous JA levels are modulated by methyl jasmonate and the JA carboxyl methyltransferase that lead to high levels in white fruit and a decline during ripening, antagonistically to ABA . In strawberry, JA appears to be involved in defence responses against B. cinerea. For example, strawberries treated with MeJA had a delayed and much slower progression of B. cinerea infections .As indicated previously, B. cinerea releases enzymes and metabolites that act as virulence factors but may also induce plant responses that are beneficial for fungal infection . A relevant example of the manipulation of physiological processes in the host by B. cinerea is the interference with specific developmental processes. In tomato plants, B. cinerea infections modified host gene expression to increase susceptibility, such as the induction of senescence in leaves . Moreover, infected unripe tomato fruit show premature expression of genes involved in ethylene synthesis during tomato ripening . These findings suggest that B. cinerea could initiate ethylene production and thereby stimulate early ripening. As strawberries are non-climacteric fruit, ethylene production of B. cinerea may not have substantial effects on strawberry ripening; however,large plastic pots the fungus was also shown to induce genes involved in the biosynthesis of other plant hormones such as ABA. Moreover, B. cinerea can synthesize and secrete ABA that functions as a virulence factor .
Besides hormones, increased oxidative reactions caused by the pathogen may influence ripening progression .Defence mechanisms can be divided into preformed and induced defences. In strawberries, preformed defence compounds are especially abundant in the unripe stage, as reviewed in the section on quiescence of B. cinerea. Even though plants accumulate defence compounds, B. cinerea has mechanisms to cope with these metabolites by efflux and detoxification of inhibitory substances. ATP-binding cassette transporters are used by B. cinerea to facilitate the efflux of antifungal compounds, such as stilbenes . B. cinerea is capable of detoxifying inhibitory substances, like epicatechin by secretion of laccases . Active B. cinerea infections can result in a reduction of specific secondary metabolites. It has been reported that levels of flavan-3-ol, benzoic acid and phenylpropanoids drop in B. cinerea-infected strawberries . Strawberries respond to B. cinerea infection by triggering defences. In some cases, preformed and induced defences can overlap such as in the case of PGIPs. An endogenous PGIP appears to be constitutively expressed in fruit from various strawberry cultivars . However, this PGIP and six additional ones show higher expression levels upon infection with B. cinerea . Overexpression of FaPGIP1a and FaPGIP2a in cisgenic plants conferred enhanced resistance to grey mould . Other enzymes induced by B. cinerea infections are chitinases. Expression of the chitinases FaChi2-1 and FaChi2-2 peaked 16 hpi in B. cinerea-infected strawberries . Furthermore, heterologous expression of Phaseolus vulgaris chitinase cH5B in strawberry resulted in higher resistance to infection . Another study demonstrated that application of heat-inactivated cells of the yeast Aureobasidium pullulans promoted tolerance to B. cinerea in strawberries . This primed resistance is probably due to the fruit’s perception of chitin from the yeast leading to induction of chitinases or other plant immune responses. Moreover, fruit defence responses may be primed using mechanical stimulation as it was reported for strawberry leaves . Induced defences include accumulation of secondary metabolites and ROS. For instance, strawberries accumulate proanthocyanins around infection zones possibly to restrict fungal growth . The surroundings of infection sites generally display higher ROS production . ROS can serve as an effective defence against pathogens but also can lead to cell death, which is considered beneficial for necrotrophic fungi . B. cinerea itself produces ROS to induce host cell death, deplete plant antioxidants and increase lipid peroxidation . It is therefore interesting that, in unripe tomato fruit ROS production leads to resistance against B. cinerea, whereas in ripe fruit it seems to promote susceptibility . Future research will likely shed more light on the role of ROS in induced defences of strawberry fruit.
Basal immunity is activated upon fungal infection. Degradation of fruit cell wall pectins can produce demethylated oligogalacturonides that trigger basal immune responses . Expression of the F. x ananassa pectin methylesterase 1 FaPE1 in Fragaria vesca resulted in reduced methyl-esterification of oligogalacturonides in fruit. This reduced esterification activated basal defences via the salicylic acid signalling pathway that led to a higher resistance to B. cinerea . Involvement of SA signalling in responses against B. cinerea was previously suggested when strawberry plants and fruit treated with SA showed decreased post harvest decay . B. cinerea can suppress the expression of plant defence responses by hijacking the host sRNA regulatory pathways . In strawberry fruits, B. cinerea infections can alter the expression of microRNAs involved in the regulation of defence genes, including the plant intracellular Ras group-related LRR protein 9-like gene . Interestingly, B. cinerea can also take up plant sRNAs during its interaction with the host. For instance, transgenic plants expressing sRNA that targets B. cinerea DCL1 and DCL2 show significantly reduced fungal growth in strawberries . The suppression of fungal growth via host sRNA is not well understood, and it is yet to be demonstrated that this mechanism of defence naturally occurs in plants.The diverse arsenal of infection mechanisms employed by B. cinerea explains its extremely wide-host range. It is therefore not surprising that entirely resistant strawberry genotypes do not exist . Several authors have analysed field resistance of strawberries to B. cinerea by quantifying disease development without artificial inoculation. A multi-year study of three strawberry cultivars found a significant effect of year, cultivar and cultivar by year interaction on the incidence of B. cinerea infections . Moreover, there was a positive correlation between row density and disease. Other studies investigated field resistance in annual winter production systems and found that variation of B. cinerea incidence between years was larger than genotype differences within years . Even though field resistance assessments investigate conditions similar to commercial production, considerable variability between environmental conditions and years can interfere with the detection of genotype differences.Due to the confounding effects of different non-genetic variables in field studies,squre planter pots assessment of post harvest resistance to B. cinerea infections has been pursued to determine genotype differences between strawberry cultivars or species. A large study of grey mould development during post harvest storage of non-inoculated fruit reported variation in disease incidence and speed of progression amongst cultivars, but no complete resistance was observed .
Another approach to reducing environmental effects in disease tests is to inoculate fruit with B. cinerea conidia suspensions. Bestfleisch et al. tested quantitative resistance in 107 accessions of wild and cultivated strawberry. In this study, two wild ecotypes of F. virginiana showed high resistance to B. cinerea infections and slow disease progression. Such high tolerance in wild species was also reported in B. cinerea-inoculated leaves and fruit of F. chiloensis accessions from Chile . In these wild accessions, B. cinerea grew much slower. Comparative studies of disease progression indicated that fruit from the cultivar Chandler developed lesions at 24 hpi, while fruit from an F. chiloensis ecotype developed symptoms at 72 hpi . Fruit were entirely covered with mould at 6 days post-infection for the cultivar Chandler and at 9 dpi for the F. chiloensis ecotype. Considering that some accessions, particularly wild ecotypes, show reduced grey mould incidence and progression, there might be genetic sources of resistance against B. cinerea that could be used to increase resistance in strawberry. However, information about resistance mechanisms is mostly based on assumptions or empirical data. Differences in ripening patterns have been suggested as a potential explanation for resistance. For instance, some strawberries ripen from inside to outside, leaving the skin, which is the entry point of infections, unripe and thus resistant for a longer time . Some more tolerant cultivars remain white or unripe around the calyx , which is where many B. cinerea infections tend to initiate. Another mechanism of resistance could be the presence of fungal inhibitors or the induction of PR proteins. FcPR5 and FcPR10 are highly induced in resistant F. chiloensis accessions when compared to commercial F. x ananassa cultivars . Based on sequence homology, FcPR5 probably possesses anti-fungal activity, and FcPR10 is likely a ribonuclease. These findings reflect that even though efforts have been made to explore resistance mechanisms of strawberry to B. cinerea, very little is known. Therefore, more research is necessary to better understand the biology of strawberry interactions with B. cinerea infections using diverse germplasm accessions.Many disease management strategies have been implemented for the control of B. cinerea in strawberry as further described below. However, even combined approaches are only capable of reducing disease incidence and severity but cannot completely prevent or eliminate grey mould in strawberries .Historically, B. cinerea infections in strawberry production have been managed by agronomic and horticultural practices, such as removal of senescent plant material to avoid inoculum buildup . Preventing contact of fruit with soil is another common practice to avoid B. cinerea infections, as most of the inoculum is present on the ground and soil moisture promotes conidia germination . Selecting the right irrigation system could help reduce grey mould incidence; mainly, the use of drip irrigation and micro-sprinklers results in limited inoculum spread and reduction of water films on the fruit . As canopy characteristics influence microclimates , nitrogen fertilization can lead to dense canopies and favour grey mould .
Similarly, shorter plant spacings promote higher incidence of B. cinerea in the field . Additionally, plastic tunnels can avoid airborne inoculum and B. cinerea incidence is lower in non-fungicide treated tunnels than in fungicide treated fields , but tunnels favour powdery mildew and complicate harvest. In summary, cultural practices are essential to limit preharvest B. cinerea infections of strawberries, especially in organic agriculture.In modern production, pesticide applications are the most common management practice for B. cinerea control . In the previous two decades, the main pesticides used in strawberry production against B. cinerea belonged to the Fungicide Resistance Action Committee Groups 1 and 2, as well as captan . However, due to increasing fungicide resistance and new legal restrictions, producers have been forced to diversify their fungicide regimen . The frequency and timing of fungicide applications are crucial for B. cinerea control. One application of fenhexamid at anthesis can be as efficient as multiple weekly applications . Additionally, alternation and combination of different fungicides with different modes of action are recommended . Resistance of B. cinerea to fungicides is a real challenge in horticulture and fungicide resistance profiles can shift considerably even within a single season . A screen of 13 B. cinerea isolates in Louisiana showed that all were partial to full resistance to FRAC 1 fungicides, and several of the isolates also had different levels of resistance to FRAC 2 fungicides . A larger survey of 1890 B. cinerea isolates revealed that 7 isolates from different locations were resistant to all single-action site FRAC fungicides groups that are registered for B. cinerea control . B. cinerea resistance to fungicides is usually associated with over expression of efflux transporters or with modification of fungicide targets. These resistance mechanisms are acquired via mutations and recombination that occur frequently in B. cinerea due to heterokaryosis, sexual reproduction and the presence of abundant transposable elements in its genome . Efflux of fungicides or accumulation of altered fungicide targets has also been shown to lead to multi-resistances . The presence of resistant isolates against the most common multi-action site fungicides reinforces the need for innovative management practices. A new generation of RNA-based fungicides has been proposed, which relies on the application of sRNA or dsRNAs that target B. cinerea virulence genes to reduce fungal infections in strawberries . However, these RNA-based fungicides remain far from commercialization, which is why fungicide resistance management such as mixture and rotation of different fungicides or testing local isolates for resistance is necessary .