1. Introduction
Fungal diseases affect crop production in most winemaking regions worldwide. Grey mould in grapevine, caused by
Botrytis cinerea, is the third most reported disease in the European, North American and Australian vineyards, and the first cause of infection in South America, where the vine is irrigated [
1]. The specific climatic conditions in the northwest Spain region, characterized by high relative humidity and moderate temperatures during the grapevine vegetative cycle favour the development of this fungal pathogen. Additionally, this fungus has a wide humidity and temperature tolerance range, which together to its ability to survive in the dry leaves and mummified grapes of the previous season enhance a constant and predominant presence of
B. cinerea in many winegrowing regions [
2,
3,
4].
Besides meteorological conditions and the presence of the pathogen, a susceptible host stage is required for the development of a fungal plant disease. This necessary condition known as disease triangle is one of the paradigms of plant pathology and supposes a valuable knowledge for Integrated Pest Management strategies, since plant disease can be prevented with the absence of any one of these factors [
5,
6]. Therefore, the analysis of airborne fungal propagules, meteorological conditions and crop phenology can be used for the determination of an optimal fungicide application schedule, which increases its effectiveness and improve the vineyard phytosanitary conditions.
B. cinerea infection can develop on grapevine at any phenological stage. However, flowering and ripening of berries stages may be more susceptible to infection due to the presence of pollen and sugary exudates at flowering that enhance the colonization of plant tissues by the pathogen, and the increase in sugar concentration in the plant that takes place during the ripening stage [
7,
8]. Moreover, infection of floral debris can act as inoculum sources for late infections that affect during the ripening of berries stage leading to important yield losses [
9,
10,
11].
Airborne fungal spore concentrations have been used as biological sensors of the pathogen’s presence and the identification of infection cycles, providing a useful tool to an early disease detection before the appearance of symptoms on the crop [
12,
13,
14]. Previous studies found a significant correlation between
Botrytis squamosa aerial conidia at a given date and lesion density one week later in onion crops, both for managed and unmanaged sites, which shows the relation of disease development with spore concentrations during previous periods [
15]. Furthermore, spores can be used for the evaluation of infection risk periods by the establishment of disease thresholds as a function of the airborne spore levels, distinguishing high-risk periods from low or moderate infection risk moments [
16]. Airborne conidia are spread units designed for fungal propagation under favourable conditions that can indicate the disease potential. Besides this valuable information, in the case of
B. cinerea we can take into account the real infective ability by immunodetection. This property can be assessed due to the specific antibodies designed for this pathogen react with a glycoprotein of the spore germinative tube or the hyphae cell wall, but not directly on conidia [
17,
18].
In the present study, we applied immunological techniques for the detection and quantification of B. cinerea conidia germinative material, in order to provide epidemiological data and complement disease detection methods based on the combination of aerobiological, phenological and meteorological data. To achieve this objective, we developed a standard curve from fresh hoovered B. cinerea conidia and a specific extraction protocol for the optimization of protein detection in aerobiological samples from a cyclone-type trap. The obtained protein concentrations were related with B. cinerea spore levels and the meteorological parameters to determine the main influential factors on this variable.
4. Discussion
The identification of new biosensors to detect the risk of plant diseases is a subject of special importance at present time, since the changes on the global climate system have also a significant impact on the distribution of crop pest and diseases. An alteration of their frequency and severity with a high variability, depending on the crop-pest system and the geographical area, is projected. Impacts caused by climate change on agroecosystems were assessed, studying a wide range of regions and crops. These studies show that negative impacts on crop yields was more common than positive impacts [
33]. Among the different crop species, the vine was widely studied in relation to climate change due to its high sensitivity to climatic conditions, with a special interest since their cultivation areas are enclosed in Mediterranean climate regions, which are important biodiversity sources at global scale [
34,
35,
36]. The temperature increase is expected to promote higher development and reproductive rates of plant pathogens, therefore an increase in crop disease severity is estimated [
37]. Additionally, variations in rainfall occurrence and their intensity, can favour fungal and bacterial species since infection, sporulation and dispersal processes of many of these species depends on humidity, increasing their occurrence as humidity periods increase [
38,
39]. Different crop-disease combinations could become problematic depending on the considered area, increasing the failure risk of current and future crop-protection strategies [
37,
40]. The prediction of crop-pest relationship evolution is an essential issue to achieve a stable crop production in the future based on sustainable strategies for crop and disease management [
40].
Several warning systems for
B. cinerea have been developed for some crop systems, based on conditions highly conducive to conidia germination and host penetration for disease development [
41]. Current knowledge on conidia behaviour is mainly based on interpreting germinative growth during artificial inoculation, in which the plants are sprayed with conidia suspensions, supplemented in some cases with nutrients to increase the possibility of tissue penetration otherwise resistant to infection [
42]. The present study provides valuable information about germination process of this pathogen in field conditions. Our study allows determining the influence of the main environmental factors and supplies the use of airborne protein concentrations as an indicator of the disease development stage and severity of fungal outbreak. The combination of immunological techniques and aerobiological monitoring improves the disease detection limit, increasing the sensitivity towards
B. cinerea fungal material, what supposes an improvement of the effectiveness of epidemiological models based on meteorological and airborne conidia concentrations.
We observed the strong influence of meteorological conditions on fungal pathogen development, since we observed variations on
B. cinerea airborne conidia related with weather conditions. The highest daily
B. cinerea airborne conidia concentrations were recorded during periods with favourable temperature and humidity conditions for the fungus in both years. These optimum conditions were detected during a temperature-increase period that ranged the optimum 20–22 °C mean temperature, considered as optimal temperature for fungal sporulation, conidia germination and infection development [
43,
44,
45]. Furthermore, the fungus wetness requirement of relative humidity higher than 95% [
46] was also reached during this period, since the high conidia concentrations were recorded just after a continuous and abundant rain period. In the case of 2018, we also detected the highest
B. cinerea conidia concentrations coinciding with favourable conditions for fungus development. During the two identified peak periods, we recorded mean temperature values around 20 °C, and a previous period of continuous rainfall that could increase the relative humidity levels, reaching the fungus wetness requirement. In addition to meteorological conditions favourable for plant infection and the presence of the pathogen, a susceptible phenological stage of the host is required for the occurrence of any plant disease. According to this, we considered the flowering (S6—BBCH scale) as one of the most vulnerable grapevine phenological stages to
B. cinerea infection. For the Ribeiro PDO vineyard area, González-Fernández et al. [
16] previously found that the highest airborne conidia levels are usually recorded near to the flowering phenological stage, thus indicating a higher activity and fungal development in this period. The bioclimatic indicator developed for this pathogen based on the Magarey model, gave as a result that the flowering stage is one of the most susceptible stages to infection [
16].
Considering the applied statistical analysis, the PCA applied on 2017 data correlated in the first component the airborne protein concentrations with mean and maximum temperatures. This relation shows the influence of temperature on the germination of
B. cinerea conidia, which is a complex process with specific requirements to occur, within a range of temperature and relative humidity [
44]. Our study detected that raises in protein levels coincided with the increase in airborne conidia concentrations as well as an increase of mean temperature in 2017. Chen and Hsieh [
47] found a thermal range for germination of
B. cinerea conidia of 16 to 28 °C, what agree with Latorre and Rioja [
48] who pointed out an optimum temperature of 20 °C for conidia germination. Similar values were found in the studied vineyard, with and average mean temperature of 18.13 °C and an average maximum temperature of 25.71 °C during the entire considered flowering period in 2017. In the third component, the 2017 PCA grouped the
B. cinerea conidia concentrations together with wind speed, which reflects the dispersion effect of wind on
Botrytis species propagules, being the predominant dispersal mechanism of these dry conidia [
42]. The 2018 PCA indicated a statistical relationship between airborne conidia and protein concentrations, grouped in the third component with an inverse relation. This correlation could reflect the inhibitory effect of high conidia concentrations on conidia germination reported for
B. cinerea and other fungal species [
49,
50,
51]. Scharrock et al. [
52] reported the production of inhibitors for
B. cinerea fungal growth and germination at concentrations up to 1 × 10
6 conidia/mL threshold as part of the self-inhibition strategy. We observed this effect during the development of the standard curve in the first point of highest conidia concentration, with a value of 6.79 × 10
5 conidia/mL. The obtained equation of logarithmic adjust shows this inhibitory effect, since the highest conidia concentration values didn’t generate a notable increase in absorbance from a certain value, tending to the curved part of the logarithmic function (
Figure 3). Nassr and Barakat [
31] also found similar results in their study of different factors affecting
B. cinerea conidia germination, since they observed the formation of conidia clots and loss of germination ability at concentrations above 4 × 10
5 conidia/mL. Regarding the Spearman’s correlation test, the correlations found for airborne conidia were mainly with temperature variables and wind speed of the 5-6-7 days before, and water-related variables (relative humidity and rainfall) of the 2-3-4 days before. These findings reinforce the obtained results by means of the PCA analysis, and agree with the stated by other authors who pointed out the seven-days period as required for fungal sporulation [
15,
53]. In the case of the protein concentrations, we found that most of significant correlations coincided on the 3 to 4 days before, mainly of mean, maximum and minimum temperatures, and dew point. We also found a strong correlation with relative humidity of the same day. This period of three to four days agrees with the indicated period by González-Fernández et al. [
16], who found a period between four to six days (depending on the phenological stage under propitious meteorological conditions) for conidia germination and plant infection since the detection of conidia presence.
In many patho-systems, infection develops in the presence of a film of water on susceptible plant tissues. The role of water and nutrients in germination have been long recognized, however the pathogen is also able to infect plant tissues without a film of water on plant surfaces [
41,
54,
55]. Several authors pointed out that
B. cinerea infections occur under high relative humidity conditions with values higher than 90% [
43,
46,
56,
57]. This humidity requirement can be met due to the occurrence of rain, drizzle or mist that can produce condensation on vine bunches or other plant surfaces. Moreover, water condensation because of evapotranspiration can be enough to initiate conidia germination and plant infection under field conditions [
48]. In addition to the role of water, exogenous nutrients are a key factor required for conidia germination and the subsequent infection process [
41]. During flowering, the pathogen uses pollen and sugary compounds produced by the plant for conidia germination, such as sugary stigmatic fluids secreted for the growth of the pollen tube [
23,
42]. The negative statistical influence of water-related variables on germinative protein concentrations detected in the regression model could be explained by the relevance of nutrient’s presence for conidia germination, since condensed water can produce a dilution or wash of nutrients in sugary solutions from plant surfaces. Elad and Yunis [
58] demonstrated that nutrients enhance
B. cinerea conidia germination, since they found that washing off nutrients led to a decrease in disease incidence. They found that no infection occurred when cucumber fruits were inoculated with
B. cinerea conidia and no nutrients were applied, as well as a reduction of 21% infection in petal-bearing fruits that were washed before inoculation with respect to those which were not washed.