Survey and prevalence of species causing Alternaria leaf spots on brassica species in Pernambuco

Brassicaceae family comprises plant species that are very important as vegetable crops, such as the species complex Brassica oleracea and Brassica rapa. Alternaria brassicicola and A. brassicae are among the most important pathogens of Brassicaceae causing Alternaria leaf spot disease. The occurrence and prevalence of Alternaria species causing leaf spots in brassica crops in Pernambuco was acessed, as well as the existence of a possible preference by vegetable host for these pathogens. Twenty-eight fields were surveyed in the Agreste region of Pernambuco state, in the 2005 and 2006 growing seasons. In each year, 10 Chinese cabbage, six cabbage, six cauliflower and six broccoli fields were visited. In each field, 50 leaves showing at least five lesions were randomly collected. Species identification was performed taking into account morphology of the conidia that was compared with literature data. Among the two Alternaria species found, A. brassicae was found in all Chinese cabbage fields while A. brassicicola was found in all fields of cabbage, cauliflower and broccoli. Overall, A. brassicicola was more prevalent than A. brassicae. In Chinese cabbage there was predominance of A. brassicae, with mean prevalence of 91.0% and 96.5% in 2005 and 2006. On the other hand, in broccoli and cabbage there was high predominance of A. brassicicola, with mean prevalence between 95.1% and 99.8%. In cauliflower, although the prevalence has been of A. brassicicola, high frequency of A. brassicae was noted. The frequency of co-occurrence of both Alternaria species was very low. The results of this study reinforce the hypothesis of existence of host preference within species of Alternaria that cause leaf spots in brassica crops, especially when Chinese cabbage, broccoli and cabbage are considered. This information is critical to developing strategies for managing Alternaria leaf spots in Brassicaceae species.

B rassicaceae family comprises lots of plant species which are vegetables of great economic importance (D'antuono et al., 2007). Among them, the most cultivated ones in Brazil are classified in the European species (Brassica oleracea), which includes intensively cultivated subspecies, like cabbage (B. oleracea L. var. capitata), green collards, (B. oleracea L. var. acephala), cauliflower (B. oleracea L. var. botrytis) and broccoli (B. oleracea L. var. italica).
Another widely cultivated species in the world and in Brazil is Brassica rapa (Brassica campestris), which complex comprises the eastern group among cultivated Brassicaceae. This complex species is also subdivided in subspecies,

Palavras
The disease can show up in nursery phase, causing the damping-off in seedlings and necrosis of cotyledons and hypocotyls. In adult plants, initially, the symptoms occur in outer leaves, developing for the whole plant, subsequently. The lesions are small and necrotic. In its development, the lesions become circular, concentric and with a chlorotic halo, usually recovered by conidia, asexual phase structures of the fungus (Maringoni, 2005).
The spots caused by A. brassicicola in the leaves are usually smaller than those spots caused by A. brassicae, and they present darker color, almost black. Typical symptoms of A. brassicicola occur in floral parts, mainly in cauliflower. In Chinese cabbage coalescence of lesions can occur, providing to outer leaves a burned look, which leads to a great depreciation of the product. Seeds in phase of formation, when colonized by fungi, can be destroyed or become empty. Mature seeds, when colonized, carry the mycelium of the pathogen externally and/or internally (Maringoni, 2005).
Alternaria epidemic occur under high relative humidity condition (>87%) and high temperatures (A. brassicicola) or moderate (A. brassicae). For the infection, at least, 9 h of free water in the surface of the plant is necessary (Mehta et al., 2005). The symptoms appear between two and 14 days after the infection, depending on the susceptibility of the cultivar and the environmental conditions. The spread of inoculums occurs through the infected seeds and seedlings and through the wind. The main source of Alternaria inoculum are contaminated seeds, rests of infected crops, weeds and spontaneous host (Maringoni, 2005;Mehta et al., 2005).
Despite the importance of the disease, there are few studies about the occurrence and distribution of the species of Alternaria in Brazil and their possible preference for particular host. Therefore, this work aimed to study, identify and quantify the prevalence of Alternaria species which cause disease in brassicae crops in Pernambuco, as well as a possible preference for host.

MATERIAL AND METHODS
Study area and sampling -In 2005 and 2006, twenty-eight brassicae fields were visited, randomly chosen, and located in four municipalities in the Agreste Region of Pernambuco state: Camocim de São Félix, Bezerros, Gravatá and Chã Grande. The visits were taken place from September to December period, at temperature of 28.2±4.6 0 C and relative humidity of 76.5±7.3%. In each year, 10 Chinese cabbage fields were sampled (cvs. Komachi and Taibyo) and six of each of the species of the following subspecies of B. oleracea: broccoli (cv. Piracicaba Precoce), cauliflower (cv. Verona) and cabbage (cvs. Fuyutoyo and Matsukaze). The plants aged between 45 and 60 days after transplanting. In each field, fifty leaves showing symptoms of Alternaria leaf spot disease were collected randomly, with at least five lesions in the leaf blade. From each of the five lesions per leaf, fragments of tissues containing fungi structures were taken out and microscope preparations on glass slides containing a drop of blue Aman were made. The slides were, afterwards, observed in optical microscope at 400x magnification.
Identification of Alternaria species -The identification of Alternaria was carried out taking in account the characteristics of the conidia, like size and chain or isolated formation. In cases of doubt, microcultures were prepared in order to confirm the species, through cultivation technique on the blade (Sutton, 1980), using block (1 cm 2 ) potato-dextrose-agar medium culture (PDA) put in the center of a sterilized glass blade, deposition of small pieces of mycelium on each side of the block (1 cm 2 ), block cover with sterilized coverslips and incubated at 25±1 0 C. After 72 hours, the coverslip was removed and put on a glass blade containing a drop of blue Aman. The glass blades were observed as described previously. The characteristics of the observed isolates were compared to the characteristics of the Alternaria species described in literature (Ellis, 1971;Simmons, 2007).
Data analysis and summarization -The frequencies observed in each of the Alternaria species and their combination within years were compared using Kruskal-Wallis test (p= 0.05). The frequency in each species and the combination of them during these years were compared using Mann-Whitney test (p= 0.05).

RESULTS AND DISCUSSION
The presence of A. brassicicola and A. brassicae was confirmed in all sampled brassicae in Pernambuco, whereas the presence of A. japonica was not recorded. These results confirm the recent observations of Reis & Boiteux (2010) about the predominance of the two species in cultivation of brassicae in Brazil. Alternaria brassicae was observed in all fields of Chinese cabbage sampled, in 66.7% of the fields of Chinese cabbage and in 16.7% of cabbage and broccoli fields. On the other hand, A. brassicicola was found in all sampled cabbage, cauliflower and broccoli fields, and in 50% of Chinese cabbage fields. Therefore, it is possible to say that, apparently, A. brassicae species prefer the Chinese cabbage host (B. rapa) and A. brassicicola species prefers B. oleracea complex hosts. Similar results were obtained, previously, with these pathosystems in Pernambuco (Peruch et al., 2006) and several regions in Brazil, including Pernambuco (Reis & Boiteux, 2010), which were observed higher frequency of A. brassicicola infecting B. oleracea complex species and A. brassicae infecting Chinese-cabbage and other species of eastern group.
When considered the two Alternaria species together, independently on the species of the host, A. brassicicola appeared in a higher frequency in evaluated lesions in two years of sampling, at average of 52.7% and 57.1% for 2005 and 2006, respectively. The average frequency of this species differed significantly from the observed for A. brassicae, which was 39.2% and 42.4% for 2005 and 2006, respectively. The presence of the two species in the same lesion was little observed in the year of 2005 (8.5% of lesions) and hardly ever in the year of 2006 (only 0.5%) (Figure 1). This difference in frequency in which the two species were found in the evaluated samples may happen because of the higher number of fields of the species B. oleracea (18) in relation to the number of fields of the species B. rapa (10) sampled in each year. However, the preference of A. brassicae for Chinese cabbage and A. brassicicola for brassicae of the European group seems to be clear in the studied region. Furthermore, as the study represents only two years, this tendency may change in the following years, depending mainly on the climate changes and the introduction of new cultivars of brassicae in the region.
When considered the isolated species of brassicae, Chinese cabbage showed higher number of A. brassicae, with average prevalence of 91.0% and 96.5% for 2005 and 2006, being significantly superior when compared to A. brassicicola or the combination of both (Table 1). On the other hand, A. brassicicola predominated in other brassicae, with prevalence of 99.8% and 99.3% in broccoli, and 95.1% and 99.5% in cabbage and 61. 6% and 64.2% in cauliflower, in 2005 and 2006, respectively. The frequency of A. brassicae in cauliflower reached 29.7% and 32.8% in 2005 and 2006, but it was significantly lower than the observed results for A. brassicicola (Table 1).
According to the results observed in this study, we can say that different   (2010) observed that isolates of A. brassicae and A. brassicicola were able to infect and cause some level of symptoms in brassicae of european and oriental groups, as well as some brassicae weeds. However, different levels of quantitative resistance in some hosts were also observed and characterized, mainly for the presence of lesions in smaller sizes.
As observed in other studies (Verma & Saharan, 1994;Peruch et al., 2006;Reis & Boiteux, 2010), the results in this work reinforce the evidence that among the species of Alternaria which cause diseases in brassicae in Pernambuco, preference for host occurs, mainly in Chinese cabbage, broccoli and cabbage.
This aspect should be considered in the development of strategy for management of Alternaria, mainly involving the use of resistant cultivars and crop rotation with different species of brassicae, considering the species of the pathogen prevailing in each species of brassicae.