Determination on Microclimatic Conditions at Vines upon Development on Gray Mold ( Botrytis cinerea )

One of the most important plant diseases in viticulture is gray mold caused by Botrytis cinerea Pers. Fr., the anamorph of an ascomycete fungus (Botryo-tinia fuckeliana Whetzel). Locality Smilica, Kavadarci, Republic of North Macedonia, was the place where experimental fields with white varieties Smedervka and Zilavka were continuously observed. Working hypothesis was to follow development of the disease after increasing glucose over 11% until the time of the grape harvest, and microclimate was monitored at the same time. In both white varieties Smederevka and Zilavka on the control variants weren’t used botricide treatments to distinguish between the variants that were conventionally treated against B. cinerea. The aim of the research was to determine how microclimatic conditions affect the development of B. cinerea and consequently to create forecasting model for gray mold. The forecasting model for B. cinerea is based on relationship between temperature and humidity in the vines’ canopies. The aim of the research is to prevent development of B. cinerea and consequently reduce the number of chemical treatments.

The control of B. cinerea in grapes has always been a challenge especially if its adaptability to environmental factors is taken into account. Hence gray mold is a facultative saprophyte which means that in its development the saprophyte and parasitic phases are present, which are an adaptive response of the pathogen to external factors. According to [4], in the parasitic phase, the gray mold kills the living cells of the plant and the colonized them, while in the saprophytic phase it draws nutrients from the dead tissue. The appearance of the saprophytic phase on the flowering elements increases the potential inoculum for more intensive development of B. cinerea in the ripening phase of the grapes. It is quite characteristic that during the pathogenesis, in different periods of time, different tissues of the vine are infected, which serve as a bridge and eventually the infection covers the mature grapes. The gray mold always runs parallel to the ripeness of the grapes and the autumn rainy season before harvest. This pathogen manifests its destructive influence from the veraison phase (onset of ripening) to grape harvest. During the process of grape berry infection by B. cinerea, various biochemical interactions take place. These interactions have been investigated thoroughly with respect to host resistance to the fungus and involve both constitutive factors and induced ones following stress or infection [5].
In varieties with very compact bunch, during maturation, more pressure is created on the grape berries and as result at this growth it happens that one berry suppresses the other and at place of junction with peduncle a small cleavage or separation of the grape berries occurs. A drop of grape juice flows through this small opening, which is at the same time a nutrient base for the develop-  [6] B. cinerea may infect grape flowers and remain latent in these tissues until grape berries begin to mature. Infection of bunches resulting from infected floral parts and aborted berries trapped within the grape bunch ("bunch trash") is also a potential infection mechanism. The authors [7] found that 28.6% of the bunch trash recovered from within bunches at veraison was infected with B. cinerea; 95.5% of this infected material was aborted berries, 3.6% was calyptrae and the remaining material was made up of leaf, stem and tendril pieces. According to [2] studied the infection of grape flowers by B. cinerea and found that floral parts were heavily colonized after 72 h but this study did not make a link between this fungal colonization and gray mold symptoms at harvest. Based on how the gray mold infects the bunches, two models of infection of grapevine can be distinguished: 1) the spores are dispersed through the vineyard and grape berries become infected by conidia; 2) in favorable condition occurs berry to berry infection with mycelium structures. Questions still exist about these two models of epidemiological development of gray mold. The authors [8] reported that removing the leaves near the bunches could successfully control the disease. Reliable methods for prognosis of the occurrence of disease in the vineyard do not currently exist [9].
For these reasons development of B. cinerea was monitored inside of canopy microclimate condition.

Materials and Methods
The research was completed in a vineyard located at Smilica near Kavadarci Republic of North Macedonia (41˚42'71.4''N, 22˚0'10.75''E) on white grape varieties Smederevka and Zilavka. A double Guyot pruning system was applied in the vineyard. The Smederevka variety was present on an area of 1.7 ha while Zilavka variety was present on an area of 0.5 ha. The variants set in the experiment considered consisted of treated and untreated grapes by simultaneously measuring microclimatic conditions and monitoring disease. Each variant was placed in an area of two rows, and the samples were taken from the middle of the variant, to prevent any external influence. Except for the control (no treatments against grey mould), which was represented by only one row, treatments against downy mildew and powdery mildew were regularly performed, but no active substances were used which could have a side effect against grey mould. The development of the grey mould was followed during the working hypothesis. From each variant, five plants were marked, and from each plant, six bunches were selected, which were marked on the rachis (handle) of the bunches with red tape. In the control, the disease was monitored in three plants.

Variants and Calculations
The essence of the initial observation is to understand the trend of the disease.
For this purpose, in the first part of the field analysis, when the incubation pe-riod and the appearance of the first symptoms should be determined, a mathematical-statistical method was used. This method involves daily measurement of temperature and relative humidity in the habitat of the vine while the working hypothesis is in progress. Tda-daily average temperature.
The next parameter to be determined is humidity point (Hp). Where is the length of retention the dew on the plant organs of the vine expressed in hours.

Working Hypothesis
The working hypothesis or observation period of gray mold started when the bunches reaches a sugar content of more than 11% until the grape harvest. At each varieties had two variants: treated and untreated grapes. In both white varieties Smederevka and Zilavka on the control variants wasn't used botricide treatments to distinguish between the variants that were conventionally treated against B. cinerea From each variant, five plants were marked, and from each plant, six bunches were selected, which were marked on the rachis (handle) of the bunches with red tape. In the control, the disease was monitored in three plants. For that time, temperature and humidity were measured using a digital thermohydrometer in the vines habitat. The length of moisture retention was measured in hours. Humidity of less than half an hour was not taken into account. Also not considered are very low they intensity rains up to 0.2 mm/h, when we have long lasting rains of more than half an hour. Then we measured

Results
This  (Table 1). In this way, the relevance of the research study was proved by analyzing the treated and untreated grapes at the same time while monitoring the development of the disease.

Discussion
The model describes two key stages of the B. cinerea life cycle in vineyards: 1) infection of mature berries by conidia; 2) development of mycelium when it occurs berry to berry infection. In order to simplify the biological cycle of development to explain the infectious features of grey mould, the following phases are  This phase represents the interaction of the spore with the cuticle surface. Initially, hydration of conidia occurs, which typically involves weak adhesive forces, resulting from hydrophobic interactions between the host and conidial surfaces [12]. A few hours after inoculation, the conidia germination germ tubes are covered with a fibrillar-like extracellular matrix material [12]. These adhesive structures are excreted by the spore and consist of carbohydrates and proteins [13].

Conclusion and Recommendations
As a result of monitoring the microclimatic conditions for the development of B. cinerea, it can be concluded that they are an essential denominator for the development of the disease. If the incubation phase of the pathogen was completed, the infection will depend on the moment when favorable conditions occur, regardless of the interruptions in the incubation process that occur as a result of unfavorable microclimatic conditions. Deteriorated external conditions during incubation in B. cinerea can cause a resistance reaction which sometimes leads us to the erroneous conclusion that there are no conditions for the development of the disease. The insight in determining the incubation period of B. cinerea is the basis for reducing the last chemical treatments just before the grape harvest.