Identification and Characterization of Bacterial Agents Causing Moderate Damage and Souring of the Fig Fruits

In order to determine the factors of decay and sourness of fig fruits, in the summer of 2016-17, 60 leaf, fruit and stem samples from different regions of Tehran, Varamin (Qal'e No), Mazandaran (Amol, Noor and Sari), Lorestan from Iran and a branch of fig fruit sample from Italy were collected. We obtained 30 isolates from the sample. The pathogenicity of 30 isolates were confirmed by artificially inoculation using fig fruits. They were also characterized based on key phenotypic traits. All 30 isolates showed hypersensitivity reaction to tobacco, pelargonium and did not show pathogenicity to potato tubers. 16S rRNA gene of the 10 representative isolates were sequenced. Ten isolates were identified as Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Pseudomonas fulva, Brevibacterium linens, Pseudomonas fragi, Bacillus licheniformis, Bacillus paralicheniformis and Bacillus cereus based on the determined sequences. None of the isolates caused fruit rot but typical disease symptoms were observed on fig leaves and fruits. This is the first report of the presence of pathogenic bacteria on fig trees in Iran.

Apart these, various yeasts, fungi, and bacteria apparently responsible for the souring disease of the fruit of the fig. Organisms are carried into the fruit by the dried fruit beetle, Carpophilus hemipterus. It is a preharvest problem resulting from yeasts and bacteria carried into the figs by insects, especially vinegar flies, resulting in odors of alcohol or acetic acid (Crisosto and Kader, 2004;Henley, 1991;Ferguson et al., 2011). Many types of fig fruit rot were observed, from the soft, watery, fermented type to the typical dry-rot type (Caldis, 1930). The aim of this study was to identify new bacterial pathogens and some bacteria affecting fruit souring of fig trees.

Isolation and Purification of Bacteria
In the summer and late autumn of 2016, 60 plant samples including branch, leaf and fruit of infected fig trees with brown spot symptoms on leaves and rot and deformity of fruits and necrotic spots on fruit and stems were collected from different regions of Tehran, Mazandaran and Varamin, Iran. A peculiar fig samples were collected from a tree grown in Modarres hospital yard in Tehran. All samples were washed with distilled water and then sterilized with 70% alcohol for 1 minute. Small pieces from border of infected and healthy tissue were prepared and each was transferred to tubes containing 5 ml sterilized distilled water. A loopful of sample suspension was streaked on Nutrient agar (NA) and King's(KB) media. Petri plates was incubated at 25°C for 48 hours. The typical single colonies were transferred to new culture media and purified. Pathogenicity tests were fulfilled and all suspected pathogens were preserved in nutrient broth containing 15% glycerol at -20°C and in NA slants under liquid paraffin.

DNA Extraction
Bacterial DNA was extracted by simple boiling method modified after Sambrook et al.,1989(Elboutahiri et al.,2009. Bacteria were grown in agar media at 28°C for 2 days. A loopful of cells was suspended in 25µl of sterile distillated water and followed an addition of 25µl of freshly prepared lysis buffer containing 0.1 N NaOH and 0.5% SDS. The mixture was boiled in a water bath for 15 min. 200µl of TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) was added to the mixture and centrifuged at 13,000 rpm for 5 minutes. Each supernatant was transferred to a clean tube and stored at -20 °C.

Preparation of Mixture Reaction for PCR Test
The final volume of the reaction mixture was 25 μl for each tube, containing 23 μl of the reaction mixture and 1 μl of genomic DNA equivalent to100 ng for each isolate. The primer pair P1(5′-AGAGTTTGATCCTGGTCAGAACGCT-3′) and P6 (5′-TACGGCTACCTTGTTACGACTTCACCCC-3′) was used (Palacio-Bielsa et al., 2009). The reaction mix was placed on a thermocycler and subjected to PCR cycles: 94°C for 2 min, followed by 94°C for 45 seconds, annealing at 58°C for 45 seconds, extension at a temperature of 72°C for 1 minute and final extension by 35 cycles at a temperature of 72°C for 20 minutes. PCR amplified products were electrophoresed in an agarose gel (1%) and were visualized using loading buffer dye (Is it right? loading buffer dye is only a guide to adjust the time of electrophoresis. You can not see PCR product). PCR products of 10 strains were sent to Macrogen Inc. (Seoul, South Korea) for sequencing. Sequencing was done in both directions of Sense and Antisense using P1/P6 primers. Sequence similarity was determined using a BLAST search of the National Center for Biotechnology Information (NCBI).  (Schaad et al., 2001). Distilled water is used as a negative control. The negative treatments were checked daily for development of any disease symptoms.
Characterization based on pathogenicity to tobacco, pelargonium and potato tubers 1-Hypersensitivity reaction (HR): Thirty isolates, which had been confirmed the capability of causing HR on tobacco leaves, were served for HR test to pelargonium leaves. At first, a suspension of bacteria with an approximate concentration of 10 6 cfu/ml was prepared and then injected with a sterile syringe to underside of the young leaves of pelargonium. The reaction was evaluated for 24 to 48 hours (Klement, 1963).

2-Potato soft rot test:
Healthy and fresh potato tubers were washed thoroughly with tap water, surface sterilized with 70% alcohol. The tuber skin was peeled and internal tissue was cut into small slices. Each slice was placed into sterile petri dish containing about 1/8 inch of sterile water and the surface was of the tissues were inoculated with a single colony of each isolate using 10? 30? isolates. Distilled water was used as negative controls. Petri dishes were kept at 25-30°C for 24-24 hours. All rotten and decay slices were recorded (Schaad et al., 2001).

Isolation and Characterization of the Isolates
Sixty fig plant samples with different diseased and abnormal symptoms from 7 cities from Iran and one from Italy were collected (Fig. 1). Thirty bacterial isolates were selected in the first stage that were capable of causing HR on tobacco leaves and to some extent disease symptoms of fig leaf and fruit. Of these, 10 isolates with sever disease symptoms were selected for PCR analysis.

Determination of the 16S rDNA Gene Sequences
All 10 selected bacteria produced a single sharp 1500bp fragment (Figure 2). The result were indicated that the strains AM2, AM5 and AM11 by 100% of homology were belonged to Stenotrophomonas maltophilia, Bacillus paralicheniformis and Pseudomonas fragi, respectively. Likewise, the strains AM13, AM15 and AM26, AM22 and AM27 by 100% of homology were assigned as Bacillus licheniformis, Serratia marcescens, Pseudomonas aeruginosa and Bacillus cereus, respectively. Percentage of homology 96% and 84% ensured that The 16S rRNA gene sequences of the strains AM12 and AM18 were also showing 96% and 84% identity to Brevibacterium linens and Pseudomonas fulva, respectively.

Testing for Pathogenicity on Fig Fruits and Leaf
All 10 selected isolates caused various disease symptoms including necrosis and chlorosis in leaves; rot and deformity of fruits and brown spots on fruit skin and stem on fig fruits. Pseudomonas aeurginosa isolate AM22 caused the wrinkling of the fruits, as it had been seen in natural conditions. On the other hand, Serattia marcescens isolates AM15 and AM26, Brevibacterium linens isolate AM12, Stenotrophomonas maltophilia AM2 and Pseudomonas fragi isolate AM11, caused brown spots, water-soaking lesions and fruit spoilage. The three more isolates namely Bacillus licheniformis AM13, Pseudomonas fulva AM18 and Bacillus paralicheniformis AM5 could cause brown spots and water-soaking lesions without fruit spoilage (Fig.3).
The four bacteria namely, Pseudomonas fragi, Brevibacterium linens, Serratia marcescens, Pseudomonas aeruginosa could have severe symptoms of the disease on Adriatic fruits rather than desert king fig fruits.

Hypersensitivity Reaction
All 30 isolates tested were able to cause hypersensitivity and necrosis on pelargonium leaves (Fig. 4).

2-Potato soft test:
None of the 30 isolates that showed necrosis on the pelargonium leaves were able to soft rot on the potato slices.

Identification of Bacterial Isolates
The ten selected bacteria were identified based on certain morphological, physiological and biochemical tests (Table 1).  You identified based on sequence similarity.

Discussion
Despite the importance of fig for Iran, which is economically valuable, the bacterial diseases of the fig trees, has remained silent until now. Perhaps one of the reasons is that there are a few reports of bacterial diseases on this host worldwide. Most diseases are caused by fungi and most prevalent fruit infection is souring which has been caused by various yeasts, fungi, and bacteria, carried into the fruit by the dried fruit beetle, Carpophilus hemipterus (Cladis,1930;Crisosto and Kader, 2004).
In this research, we obtained 60 fig fruits isolates from seven cities in Iran and one city in Italy.
The distribution of these bacteria and the areas where these isolates were isolated are as follows: P. aeruginosa and S. marcescens were isolated from leaf and fruits of the According to numerous reports, these bacteria cause an infection in humans, especially in patients with immune deficiency (Claus,1986;Aslani, 2009). It was reported by Kamar, 1989 that Bacillus cereus is the cause of two types of poisoning (diarrhea and vomiting). In the case of Brevibacterium linens and Pseudomonas fragi, these two species have been isolated from plant and dairy products and have been caused the consumer illness (Pereira, 1957). Whereas, Pseudomonas fragi was isolated from lettuce (Edelman and Lin, 2014). Pseudomonas aeruginosa, a common opportunistic bacterium between humans and plants was isolated from diferent sources such as rhizosphere of corn (Tahmasebi et al., 2014) and tobacco leaf in China (Yu, 2008). Another important species was Pseudomonas fulva, a clinical isolate which has been reported mostly from aquatic environments including rice seed and fennel plants (Edelman and Lin, 2014). Serattia marcescens, a ubiquitous bacterium has been isolated from plant materials such as watermelon (Hosseini et al., 2013) and nutty trees (Habibi and Khodaygan, 2013). The latter, was first isolated from figs in New York (Edelman and Lin, 2014).
Bacillus licheniformis, has a wide range of hosts and is isolated from soil containing vegetable waste (Nagaraju, 2013), wheat fields of central provinces, Golestan and Mazandaran (Etebarian et al., 2008), cucumber farrowing in Jiroft (Rezaei et al., 2016), soil and water around the hot springs in Semnan (Moghbeli and Noshiri, 2009). Bacillus paralicheniformis like most of the above bacteria, has been isolated on various plants rather than the figs. For example, from soybean paste (Christopher, 2015), soil containing citrus waste soil in several regions of Golestan province (Mohammadi et al., 2016), wheat fields of central provinces of Golestan and Mazandaran (Etebarian et al., 2008), rhizosphere of orange trees in Kerman province (Ramezani et al., 2013) and also from asparagus plant in Iran (Faghanizadeh et al., 2016) and in New York (Edelman and Lin, 2014).
In conclusion, it should be noted that all bacteria except Serratia marcescens are reported for the first time on fig plants in Iran. There is no doubt that water, food and vegetables play an important role in the spread of pathogens and secondary infections. Given that some of these causal agents are transmitted through consumption of fresh fruits and vegetable, planting fruit trees in open gardens of hospitals is a major mistake and potentially very dangerous for the incidence and spread of diseases with a common human-plant hosts. And finally, due to the abundance of microbial populations in soft rot and damaged fruits, their consumption should be avoided to maintain the health of the body.