Fungal Biodiversity of Strawberry Fields in Aydin , TURKEY

Strawberry is a kind of delicious and aromatic fruit, which can be consumed as fresh and also is suitable for industry. However, strawberry is exposed to many fungal diseases. The aim of this study is to determine the fungi that present in the field whether or not pathogenic. Samples were collected from different strawberry fields in Aydın in April 2015. Morphological identification was made according to the shape and color of the colonies, mycelium and spore structures. For molecular identification, ITS rDNA gene region was used. According to morphological and molecular methods, eleven different fungal genera were found on strawberries. ARTICLE HISTORY Received: 24 October 2017 Revised: 06 February 2018 Accepted: 13 February 2018


Introduction
Biodiversity is the foundation living systems to which human success is actually associated [1].It is one of the basic parts of nature and it ensures the survival of earth.Strawberries are known as plants belonging to the genus Fragaria.Taxonomically, the genus Fragaria is included in the family Rosaceae.Strawberry is a perennial herbaceous, short day plant.Strawberries are a consumable plant part, which also used in industry.Be that as it may, natural products are easily spoilt and as a rule have dynamic digestion amid the capacity organize [2].The significance of organic products in human nourishment can't be overestimated as it gives basic development factors, for example, vitamins and minerals important for continuation of human life [3].The high concentration of various sugars, minerals, vitamins, amino acids, and low pH also enhances the successful growth and survival of various forms of fungi [4].Annual reports have shown that 20% of fruits and vegetables produced are lost to spoilage [5].
According to Food and Agriculture Organization (FAO) (2012), Turkey was in the 3 rd place in strawberry production.But about 15% of the products were lost in the field before harvest due to the diseases according to farmers.
The organic action is a fundamental factor in the physical and substance advancement of soils [6].There are 110.000defined fungi species were present in the World but it is estimated that 1.5 million fungi species exists [7].The ITS region is considered to be a good candidate for accurate detection and can largely separate from all other species by this application.
It is essential to decide the decent variety of organisms, which cause infections on strawberries and their natural and hereditary impacts.In this study, fungi on strawberry fruits will be detected by morphological and molecular methods.

Sample Collection
Samples were collected aseptically from the strawberry fields in Aydın (Yenipazar, Atça, Sultanhisar, Köşk and Umurlu) in April 2015 during harvest time.Rotten strawberry fruits were collected in sterile plastic bags and kept in the portable refrigerator until brought to the laboratory.

Isolation of Fungal Species
One gram of strawberry fruits was weighted and homogenized in 9 ml of 0.85% physiologic Saline Water (PSW).100 µL of these homogenized samples were inoculated on Rose Bengal Choloramphenicol Agar and Potato Dextrose Agar.Samples were incubated at 27°C for 5 days.After the incubation, the differentiated fungi samples were selected and isolated from the mixed colony under the same incubation conditions.

Morphological Identification
Morphological identification of the samples was realized according to Samson [8].Mycelium and spore structures smeared on a slide, dyed with lactophenol cotton blue and visualized under the microscope.Colonial shapes were determined and used to identify species microscopically.

Molecular Identification
Fungi samples were put in 1.5 ml eppendorf tubes using a sterile toothpick.After, samples have reduced the powder using liquid nitrogen.DNA isolation of the samples was realized with 2X CTAB isolation protocol according to Doyle and Doyle [9].Concentration and purity of the samples were measured with a Nanodrop Spectrophotometer (Thermo).ITS rDNA gene region was used to identify the species (ITS1: 5'TCCGTAGGTGAACCTGCGG'3, ITS4: 5'TCCTCCGCTTATTGATATGC'3) [10].PCR reaction conditions were: initial denaturation 94 ºC 5 min, denaturation 94 ºC 30 sec, annealing 60 ºC 30 sec, extension 72 ºC 60 sec with 35 cycles and a final extension at 72ºC 10 min.Reagents concentrations were: 10X Taq Buffer, 0.5M dNTP mix, 10 pM from each primer, 7.5 mM MgCl2 and 1U Taq polymerase (ABM) with the final volume of 25 μl.Agarose gel electrophoresis of the samples was observed on 1.4% agarose concentration at 90 V 40 min.100 bp DNA ladder was used for size comparison of the products.PCR products were sent to DNA sequencing (Macrogen, Holland).

Data Analysis
Sequence results were aligned with the ones in GenBank using BLASTn software to find out the species of the samples.MEGA6 was used to infer phylogenetic tree.

Morphological Identification
Morphological methods showed eleven different fungal species (Table 1).Colony shape, mycelium and spore structures were observed to this purpose.

Molecular Identification
ITS rDNA gene region was used to identify fungal samples at the species level.PCR products were sent to sequencing to Macrogen (Holland).Molecular identification was made by comparing sequences with GenBank using BLASTn.Nine fungal species were found in contrast with morphological results (Table 2).MEGA6 was used to construct the phyloenetic tree.Maximum likelihood method based on the Jukes-Cantor model was used (Figure 3).MP tree was obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value.

Discussion
Eleven kinds of fungi differentiated from each other in morphological are identified from these strains.For molecular identification, rDNA ITS gene part of 78 examples is multiplied and as a result of the compare with GENEBANK data, 20 different kinds that belong to 9 species were determined.Literature shows that procedures, such as gathering and transporting, natural products may experience physical damage that builds post-reap misfortune and the likelihood of contagious pollution [11,12].Kasiamdari et al. (2002), isolated R. solani CFM1 isolate from cabbage, designed two primers from the ITS gene region and suggested that molecular methods would provide more accurate results than classical methods [13].Staats et al. (2004) used the DNA sequence of 3 nuclear protein-coding genes (RPB2, G3PDH and HSP60) to classify Botrytis spp.They also compared them to conventional methods.The results of phylogenetic analyses were showed that Botrytis spp.were separated from Sclerotiniaceae species [14].
Khairnar et al. (2011) investigated soil-borne fungal biodiversity of some fruit crops in India and found 21 different fungal species and suggested that all twenty one fungal species can be controlled with 500 ppm Moximate, a fungicide [15].Mailafia et al. (2017) researched fungi associated with fruit species and identified six different fungi and one yeast species [5].
Botrytis cinera is the cause of gray mold disease [16].Lichtheimia corymbifera is the principle pathogen causing human and animal infections.Though only one sample was found, it wasn't widespread in the sample location [17].Syncephalastrum monosporum is the endophytic fungal community of cacao and can also be found in the eyes of healthy horses, nests of laboratory reared leaf cutter ants, poultry feed, and spices [18].Rhizopus oryzae is commonly found on dead organic matter and cause of disease [19].Alternaria alternata is a common plant pathogen [20].

Conclusion
This study was made to detect fungal biodiversity on strawberries in Aydın, Turkey.As a result, nine fungal species were identified both by morphological and by molecular methods.Despite the usage of fungicides fungal diseases, such as gray mold, leaf spot disease can still be seen frequently both pre-and post-harvest.These species only were found on fruits of the plant.Investigation of soil and other plant parts can be resulted in more fungal species to be found.

Figure 3 .
Figure 3.The evolutionary history was inferred using the Maximum Likelihood method.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.All positions containing gaps and missing data were eliminated.Evolutionary analyses were conducted in MEGA6.Percenteces of the branches were shown on the nods.

Table 1 .
Morphological identification of the species

Table 2 .
Molecular Identification of species