Filamentous fungi isolated from the rhizosphere of melon plants ( Cucumis melo L . cv . Gold Mine ) cultivated in soil with organic amendments

(Filamentous fungi isolated from the rhizosphere of melon plants (Cucumis melo L. cv. Gold Mine) cultivated in soil with organic amendments). Rhizosphere soil samples were collected in a semiarid area, in the region of the São Francisco River valley, Petrolina, Pernambuco state, Brazil, to study the diversity of fi lamentous fungi in a soil cultivated with melon (Cucumis melo L. cv. Gold Mine) and receiving different organic amendments: Treatment 1 (control, without organic compost); T2 (77% coconut fi ber, 20% goat manure and 3% K2SO4); T3 (10% Ricinus communis leaves and stems, 50% Pennisetum purpureum leaves and 40% goat manure); T4 (77% coconut fi ber, 20% goat manure and 3% termophosphate); T5 (47% Pennisetum purpureum leaves, 50% goat manure and 3% K2SO4); and T6 (57% Pennisetum purpureum leaves, 40% goat manure and 3% termophosphate). Fungal isolation was carried out by the serial dilution technique to 1:1000. The Sorensen index of similarity, frequency and distribution of the fungi were evaluated. Seventy-eight species of fi lamentous fungi were isolated and identifi ed, plus several Basidiomycota (04) and Mycelia sterilia (02). The predominant genera were Aspergillus and Penicillium, with 15 and 13 species, respectively. A greater number of species was found in the sowing period (49), and in relation to the organic fertilization, treatment 6 provided the greatest species diversity (43 species). Most of the species are saprobes and only a few are considered to be potential pathogens on melon plants, such as Fusarium oxysporum, F. solani and Myrothecium roridum.


Introduction
In Brazil, the culture of melon presented great evolution in the period from 1987 to 2006, with production of fruits going from 37,980 to 500,021 tons.Melon is largely cultivated and one of the most important products of the Brazilian agribusiness that is conquering greater shares in the national and international markets.The Northeast Region is responsible for about 93% of the national melon fruits production, with 465,623 tons; the States of Rio Grande do Norte, Ceará, Bahia and Pernambuco are the main producers and exporters (Araújo & Vilela 2003;IBGE 2006).
The melon (Cucumis melo L.) started to be cultivated in the region of the submedium São Francisco in 1965, in the city of Santa Maria da Boa Vista, Pernambuco State.With the implement of several public projects for irrigation, this agriculture cluster became one of the main zones of production and exportation of fruits in the country.The culture of melon has been intensifi ed in the region, mainly in the cities of Petrolina and Juazeiro, which have better infrastructure for commercialization (Oliveira 1991).
The melon culture in the Vale do São Francisco can be carried out throughout the year, due to the favorable soil and climate conditions, with increased solar radiation, high temperatures and low relative humidity.These factors favor the concentration of total soluble solids in the fruits and diminish the incidence of diseases, increasing the quality of the fruits (Dias et al. 1998).
With the advance of organic agriculture for fruit production in the Submedium São Francisco, many types of residues have been applied; however little is known about the effect of these residues in the soil mycobiota.The type of soil amendment can have a signifi cant effect on the soil mycobiota mainly to favor the saprophytic activity in detriment of potential plant pathogens.Thus, organic residues that favor the increase of natural fungal population are important for improving the nutritional condition of the cultures of interest in the region and to ensure the sustainability of the organic production.
This work aimed to isolate and identify fi lamentous fungi from the rhizosphere of melon plants fertilized with distinct organic composts, and to evaluate the infl uence of these composts in three periods (sowing, fl owering and post-harvest) of plant development, relating them to the presence of fungi.

Materials and methods
Soil sampling -Rhizosphere soil samples were collected using an auger, to a depth of 20 cm, in a semiarid area cultivated with melon (C.melo cv.Gold Mine), in the Vale do São Francisco, Petrolina, Pernambuco State, Brazil (09º32´09´´S, 40º55´28´´W), during the periods of sowing, fl owering (37 days after sowing) and post-harvest (31 days after fl owering), in the months of October, November and December/2005, respectively.
For each treatment, three samples were taken, in a total of 18 samples at each sampling period (sowing, fl owering and post-harvest).Consi-dering all treatments, a total of 54 soil samples were investigated for the presence of fungi.The soil (Gray Argisol) samples were chemically analyzed at the Soil Laboratory of the Embrapa Semi-Árido (Tab. 1) (Embrapa 1997).Isolation and identifi cation of the fi lamentous fungi -The fungi were isolated using the serial dilution technique (Mehan et al. 1991): 25 g of each soil sample were suspended in 225 ml of sterilized distilled water (SDW) (1:10); 10 ml of this suspension were added to 990 ml of SDW (1:1000).Then, 1 ml of this suspension was cultured, in triplicate, in Petri dishes containing Sabouraud Agar added of chloranphenicol (500 mg l -1 ).The plates were kept at room temperature (28ºC) and the development of colonies was observed until 120 h, when the CFU were estimated.After the fi rst 72 h the colonies were transferred to Petri dishes with agar medium (potato dextrose agar, czapeck agar and/or malt extract agar) and kept for 20 days, allowing the formation of reproductive structures.The identifi cation of the species was carried out through macroscopic and microscopic observation of the isolates, consulting the specialized literature (Raper et al. 1949;Ames 1961;Corlett 1966;Rifai 1969;Booth 1971;Ellis 1971;Nicoli & Russo 1974;Samson 1974;Arx 1975;Ellis 1976;Sigler & Carmichael 1976;Carmichael et al. 1980;Domsch et al. 1980;Sutton 1980;Schipper 1984;Arx et al. 1986;Pitt 1988;Udagawa et al. 1989;Klich & Pitt 1994;Hanlin & Menezes 1996).Statistical analyses -The experimental design was of random blocks in a factorial arrangement 3x6, with three periods of evaluation (sowing, fl owering and post-harvest) and six fertilization treatments (T1, T2, T3, T4, T5 and T6), with three replicates.The values of the CFU were square root (x + 1) transformed before the analysis of variance (ANOVA).The averages were compared by the LSD (least square deviation), 5% of probability, using the program Statistica 5.0 (Statsoft 1997).
The Sorensen index of similarity (Müller-Dombois & Ellemberg 1974) was applied to verify the similarity between the fungal populations isolated in the different sampling periods.Frequency and distribution of each fungal species were calculated for each sampling period according to Brower et. al (1990) and Schnitter & Stephenson (2000).
The variance analysis showed differences in the number of CFU between the sampling periods and between the organic fertilization treatments.A greater number of CFU was evidenced in the sowing period (183.5 x 10 4 CFU g -1 ), differing signifi cantly from the other periods: fl owering (85.05 x 10 4 CFU g -1 ) and post-harvest (100.38 x 10 4 CFU g -1 ).In relation to the organic fertilization, treatment 5 yielded a greater number of CFU, followed by treatments 3, 4 and 1 which did not differ signifi cantly.Signifi cant differences were registered only between treatments 5, 2 and 6 (Fig. 1).
There was no difference on species diversity between the sampling periods.A greater number of species was found in the sowing period (49), without signifi cant difference in comparison with the other periods: fl owering (39 species) and post-harvest (41 species).
Studying the microfungi of the rhizosphere of Zygophyllum qatarense Hadidi, in a semiarid environment of Bahrain, Mandeel (2002) reported the predominance of species of Aspergillus and Penicillium.Similarly, Grishkan et al. (2006) found species of Penicillium and Aspergillus to be the most abundant in the rhizosphere of native plants of Negev, Israel.These results are similar to the fi ndings of this work, considering that Aspergillus and Penicillium are well represented in the studied area, with 15 (18.75%) and 13 (16.25%)species, respectively.
In the Brazilian semiarid region, Silva & Cavalcanti (1990;1991) registered Fusarium and Penicillium as predominant in the rhizosphere of tomato (Lycopersicon esculentum Mill).Maia & Gibertoni (2002)  Several of the fungal species found here were reported in areas of the Brazilian semiarid region (Maia & Gibertoni 2002;Cavalcanti et al, 2006;Costa et al. 2006).
The prevalence of Aspergillus and Penicillium occurs probably because these genera have a high number of species and are capable of surviving in dry environments (Dix & Webster 1995).
Three of the species found in this study were previously regarded as pathogen to melon plants: Fusarium oxysporum, F. solani and Myrothecium roridum (Marinho et al. 2002;Muniz et al. 2004;Viana et al. 2001).However, it is not possible to say if they were in the rhizosphere as saprobe or pathogen.These species were also registered, respectively, in the rhizosphere of sugar-cane, tomato and sunfl ower (Santos & Cavalcanti 1989;Silva & Cavalcanti 1990, 1991;Souza-Motta et al. 2003).In the present work these species showed an occasional distribution.
The organic fertilization in the melon plants favored the presence of fi lamentous fungi and increased the number of CFU.It also allowed a wide distribution of saprophytic fungi, considering that 96.25% of the species isolated in this study have been described as organic matter decomposers.Therefore, the introduction of organic composts can improve the soil quality in the culture of melon by increasing the diversity and number of fungi.
The phenology of the melon plants did not affect the diversity of fi lamentous fungi; however, it signifi cantly decreased the amount of CFU.The highest number of CFU was observed in the sowing period, probably due to the processes of incorporation and mineralization of the organic residues in the soil, resulting in an increase of the cation exchange capacity (CEC) in the subsequent periods (Tab.1).Except for treatments 5 and 6, the quantity of organic matter in the soil was higher in the sowing period, increasing the offer of substrate for decomposition that was refl ected in the highest number of CFU in this period.
The analysis of similarity showed a lower index between the fungi in the sowing and the post-harvest periods.According to Melnitchouk et al. (2005) and Wellbaum et al. (1999), this may be associated with the changes in the composition of root exudates over the phenological cycle of the plant, causing variation in the rhizodeposition, and with the hydrosoluble substances released by decomposition of organic matter, which represent an energy source promptly available for the microorganisms.In addition, Parkinson et al. (1963) apud Souza-Motta et al. (2003) mentioned that the young roots are initially colonized by a diversity of soil fungi which after some days are substituted by a more restricted mycobiota that remains until the senescence of the roots.
The results attained in this study suggest that anamorphic fungi dominate the soil mycobiota of cultivated melon fi elds in the Brazilian semiarid, and that species of Aspergillus and Penicillum are the most commonly found.Furthermore, the data indicate that the composition of the rhizosphere interfere in the fungal succession.

Table 2 .
Filamentous fungi colony forming units (CFU x 10 4 g -1 ) isolated from the rizhosphere of melon plants (C.melo cv.Gold Mine), fertilized with organic composts, during the sowing, fl owering and post-harvest periods.
Cavalcanti et al. (2006)Trichoderma.Species of Aspergillus and Penicillium predominated in soil from a copper mining impacted area(Costa et al. 2006) in Bahia State.Likewise,Cavalcanti et al. (2006)registered several fi lamentous fungi in soils from the Xingó region (Sergipe and Alagoas States), with predominance of Penicillium and Aspergillus species.