Infection of rose flowers by Botrytis cinerea under different temperatures and petal wetness

The effect of temperature and petal wetness on the infection of Botrytis cinerea in rose flowers was studied by combining temperatures of 10, 15, 20 and 25°C with periods of petal wetness of 8, 16, 24 and 32 h. An increase in the severity of gray mold was observed when petal wetness period increased. Lower level of disease severity was verified at 10°C for all wetness periods as well as for the temperature of 25°C and wetness period of eight hours. In this wetness period, the lowest severity indexes were observed in all the temperatures tested. The maximum disease severity was observed at 20°C with 24 h of petal wetness. A model of multiple regression analysis was tested to associate temperature and wetness period. A quadratic effect of temperature was observed, which was overcome by free water time. The results show that infection on the rose flower petals depends on the period of wetness of the petals; maximum estimated severity occurred at 25°C with 32 h of petal wetness; the temperature of 10°C can reduce the severity of gray independently of the petal wetness period; at higher temperatures high disease severity is dependent on the wetness on the petals.


INTRODUCTION
Roses (Rosa hybrida) are affected by several diseases that cause important losses.Gray mold caused by Botrytis cinerea is one of the most important diseases of this crop.The pathogen has worldwide occurrence and infects a great number of hosts including violet, begonia, chrysanthemum, gerbera, dahlia, geranium and tulip (Agrios, 1988;Jarvis, 1977;Marrois et al., 1988;Terry and Joyce, 2004).
B. cinerea occurs in various countries worldwide where roses are cultivated.This fungus is especially important because it causes petal spots, which evolves to rot petal areas reducing flower quality and yield (Volpin and Elad, 1991).In countries with usually moderate climate it was observed that epidemics caused by B. cinerea are more likely to occur under high relative humidity and low temperatures (Phillips and Margosan, 1985;Elad and Volpin, 1991;Hammer and Marois, 1988;Agrios, 1988).At the extent of our knowledge, in Brazil , there are no *Corresponding author.E-mail: alderi.araujo@embrapa.brAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License studies about the influence of climatic conditions on gray mold incidence in rose.
Although in Minas Gerais State roses are cultivated in greenhouses, where environmental conditions may be controlled to some extent, a high incidence of gray mold has been observed during rainy seasons.This happen perhaps because temperature is normally low and relative humidity is high in this period of the year.The petals sometimes do not present symptoms during harvest.Although the petals are handled at temperatures between 2 and 10°C after harvest, higher incidence of gray mold has been observed, mainly during transport and storage.The conidia of B. cinerea have an important function in gray mold epidemics (Jarvis, 1977;Braun and Sutton, 1987;Braun andSutton, 1988, Church, 1992).Infections initiated by conidia can be completed in about eight hours, if high humidity and low temperature are available (Baker, 1946, Mackeen, 1974).However, in geranium there was an increase in the severity of gray mold from 10 to 25°C and the optimum temperature for maximum disease severity was 25°C (Hyre, 1972).
Immersion in water is probably the main requirement for conidia germination of B. cinerea (Jarvis, 1977).This fact is associated partly with low content of water in the conidia (Yarwood cited by Blakeman, 1980).The period of wetness in relation to infection of B. cinerea depends on the host.Bulger et al. (1987) reported an increase in the infection of strawberry flowers after an increase in the period of wetness, independent of the temperature.The purpose of this work was to study the influence of different temperatures associated with different periods of petal wetness on the infection of B. cinerea.

MATERIALS AND METHODS
Rose petals of the variety Kiss were collected from commercial greenhouses in Antonio Carlos county, in Minas Gerais, Brazil.To avoid the occurrence of latent infection from natural inoculum, three petals were cut around each bloom.The stems were immersed in tap water in plastic bags to a depth of 15 cm and then transported to the laboratory.The isolates of B. cinerea used in the work were cultivated in test-tubes at 20°C under 24 h fluorescent light, for 14 days (Bulger et al., 1987).The conidia were removed with distilled water using a small brush, and the inoculum concentration was adjusted to 2 x 10 5 conidia/ml.For inoculation a De Vilbiss atomizer run by compressed air was used.The inoculum suspension was sprayed on the petals until the petal surface was completely covered, without dripping.Afterwards, the bags were covered with damp plastic bags to ensure high levels of relative humidity and to maintain wetness on the plant tissue during the period to be assessed.
The bags with the rose petals were transferred to growth chambers (Fanem BOD, São Paulo, Brazil) adjusted to various temperature levels.The effects of temperature (10, 15, 20 and 25°C) and of petal wetness duration (8, 16, 24 and 32 h) on infection by B. cinerea were studied.After each period of wetness the bags with the flower were removed from the incubators, the plastic bags were removed and the surfaces of the petals were dried using an aerator.Afterwards the plastic bags were transferred to a growth chamber at 20°C.
Forty-eight hours after removing the last treatment from the chamber, the severity of gray mold was assessed in two petals per bloom using a scale based on the scales of Horsfall and Barratt (1945) and Redmond et al. (1987) in which 1 = 0%, 2 = 0-2%, 3 = 2-5%, 4 = 5-10%, 6 = 15-25%, 7 = 25-50%, 8 = 50-75%, 9= 75-100 and 10 = 100 % of area of the petals with symptoms.There were three repetitions for each treatment in which a bag with eight blooms was a repetition.A control was inoculated with distilled water for each temperature vs. wetness combination.A multiple regression model was applied to the data in which the temperature and the wetness period were considered the independent variables.

RESULTS
There was an increase in disease severity due to the association of different temperatures with increasing wetness periods.Lower severity indexes occurred at 10°C for all wetness periods.Similar results were observed at 25°C for the wetness periods of eight and 16 h.The higher severity values occurred at 24 and 32 h of wetness and maximum severity was observed at 20°C with 24 h or more of petal wetness (Figure 1a).After this period and under the same temperature, the disease severity showed a tendency to reduce.At 16 h of petal wetness disease severity increased up to 35% for the temperature of 20°C, dropping drastically to around 12% at 25°C.Similar results were estimated by linear regression analysis, where a quadratic effect of temperature on disease severity was observed with the increase in the wetness period.Results show that, starting at 15°C, the longer the wetness period the greater the severity.The maximum disease severity estimated was at 25°C after 32 h of petal wetness (Figure 1b).The lower severity indexes were estimated at 10 and 25°C and eight hours of wetness.

DISCUSSION
Lower temperatures are probably more critical to the pathogen and, at higher temperatures, there is a compensation phenomenon (Rottem, 1978) due to the increase in the wetness period.That is, at low temperatures the disease severity has a tendency to remains low, whereas at higher temperatures the reduction of the disease severity index depends on the wetness of the petals.It is possible that the pathogen may infect the flower petals in the field, before harvest, where environmental conditions are favorable.Nevertheless, these conditions have to be maintained if the pathogen needs to grow and to sporulate on the host tissues.If after field infection favorable environmental conditions are not available, the pathogen can remain latent in the tissues of the petals until conditions improve (Elad, 1988;Elad and Volpin, 1991).
The temperature of 10°C is less favorable to mycelial grow of B. cinerea than temperatures between 25 and 30°C (Araújo et al., 2005).In post-harvest handling, temperatures between 2 and 10°C can be unfavorable to B. cinerea.However, in this work, the low severity values observed at the temperature of 10°C do not necessarily represent small losses caused by gray mold after harvest.The majority of losses caused by B. cinerea occur in the packing house and during transport to the market (Elad, 1988;Hammer and Marois, 1988).Therefore, it is important to consider the latent infection, which is imperceptible during the harvest, but can develop under humid conditions in mature flowers (Volpin and Elad, 1991).In post harvest, due to low temperature in the packinghouse and in the transportation vehicle, the pathogen stays latent in the tissues of the petals.When the petals are put in bags at room temperature (20 to 25°C) the pathogen is provided with excellent conditions for renewed development, and symptoms of gray mold reappear.It is not yet known exactly how long B. cinerea can survive under latent conditions in the tissues of the host.Therefore, if it is considered that the infection normally occurs from the beginning of the opening of the sepals and that between this period and the harvest there is a period of six to eight days, it is probable that the infection stays latent for more than 10 days.Araújo et al. (2005) used infected material from greenhouses and maintained it at 4°C observing that the latent period lasted for up to 15 days after harvesting.Barners and Shaw (2002) observed that the latent infection by B. cinerea was frequently detected in young Primula x polyantha (horticultural hybrid polyanthus) plants.Genetically marked isolates were used to demonstrate that conidial inoculum applied to young plants generally did not result in disease appearing on the leaves until flowering, regardless of when the plants were inoculated.Problems with gray mold are frequently reported during storage and transportation.In this case, it is probable that prolonged periods of wetness may increase severity of the disease, even at lower temperatures.If the petals are not infected, but there are conidia on their surfaces, they may cause infection under prolonged periods of wetness, which will cause losses of the rose flowers.Under a longer wetness period it is possible to increase the range of appropriate temperature for infection by Botrytis species.Shoemaker and Lorbeer (1977) observed that infection by Botrytis squamosa on onions could occur between 9 and 23°C, when they are exposed to water for about 40 h.
The temperature and wetness period are considered important variables for the infection of onion by B. squamosa.Alderman and Lacy (1983) observed the necessity of at least six hours of exposure to water for successful infection development of leaf blight in onion.In their work fewer lesions occurred under six hours of wetness than at 20°C, but more lesions were verified under 12 or more hours of wetness at the same temperature.Similar results were published by Nelson (1951) who demonstrated that infection of grapes by B. cinerea was higher after 21 h of wetness at 12 and 24°C, and by Ramsey and Lorbeer (1986) who found that between six and 24 h of wetness at 21°C are needed for infection of onion flowers by B. squamosa, B. allii or B. cinerea as well as 12 to 48 h are necessary for flower blight.The results of the present paper show a similar tendency to previous reported works, especially in relation to a more prolonged wetness period and the ideal temperature range for infection by B. cinerea.Although gray mold maximum disease severity was observed at 20°C and 24 h of petal wetness, through the regression equation the maximum disease severity was estimated to occur at 25°C and 32 h of petal wetness.
Under higher temperatures, the flower petals have a tendency to age more quickly or to liberate exudates with more nutrients, which favor the infectiveness by conidia (Blakeman, 1980, cited by Kerssies, 1992).Therefore, anything that can reduce the physiological stress level of rose petals may result in reduced losses due to gray mold.The implementation of control measures in the field must take into account knowledge about environmental favorable conditions for infection by B. cinerea.From the results of the present work it is possible to deduce that periods of more than 8 ho with relative humidity above 90% and temperatures in the interval of 15 to 20°C, or more than 16 hours of 90% RH and temperatures higher than 15°C, are favorable to the incidence of gray mold in rose flowers.
Considering the losses induced by B. cinerea during transportation and storage, and the low disease severity at 10°C observed in the present paper, it is important to determine the capacity of the pathogen to cause damage to rose petals under lower temperatures, including its effect on latent infections.
The implementation of measures to reduce the level of stress on the petals after harvest and to minimize the effects of latent infection must be encouraged.These studies must consider post-harvest practices of control, management of temperature and humidity in the packing house and prolongation of pot life of the flowers.In field conditions, it is important to observe environmental conditions constantly, so as to identify the most favorable period for incidence of gray mold, and thus to introduce control measures as quickly as possible.

Figure 1 .
Figure 1.Effect of the temperature and wetness period on the infection of rose flower petals by Botrytis cinerea.(a) Observed and (b) estimated.