Characterization and identification of soft rot bacterial pathogens in Bangladeshi Potatoes

We reported the characterization and identification of pathogenic bacterial strains causing soft rot in potato tuber in Bangladesh under storage conditions. Identification of bacterial pathogens in tuber is important to take appropriate disease control measures and to develope resistant plants through classical breeding or genetic engineering. A total of 92 bacterial strains were extracted from the soft rotted potato and 26 of them were found to produce soft rot on potato slices under control environment. Out of 26 soft rot causing bacterial strains, 12 were found to be aggressively virulent and were selected for characterization and identification by physiological and biochemical testing schemes. Out of the 12 virulent strains, 8 isolates designated as P-01, P-04, P-11, P-48, P-111, P-134, P-138, and P-169 were identified as Erwinia subsp. carotovora and 4 isolates labeled as P-28, P-128, P-142, and P-179 were identified as Erwinia chrysanthemi.


INTRODUCTION
Potato (Solanum tuberosum) is one of the most cultivated crop plants of the temperate and warmer regions of the world.It is the fourth most abundant food crop of the world after rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum) (Czajkowski et al., 2011).Appro-ximately 22% of potatoes are lost per year due to viral, bacterial, fungal, and pest attack to potato tuber and potato plant, incurring an annual loss of over 65 million tones (Czajkowski et al., 2011).Bacteria belonging to the Pectobacterium and Dickeya genera cause soft rot to potato tuber and blackleg in potato plant and are next to Clavibacter michiganensis that causes wilt, in terms of economic importance (Baghee-Ravari et al., 2011;Czajkowski et al., 2011).
Bacterial soft rot commonly occurs in potato tuber after *Corresponding author.E-mail: eaqubali@gmail.com.
harvest and during storage.Several bacterial species under different genera produce various cell-wall degrading enzymes that allow infiltration and maceration of parenchymatous tissues of a wide range of plants on which they feed (Czajkowski et al., 2011).Although many bacteria possess ability to produce tissue macerating enzymes, only few of them such as Pectobacterium atrosepticum (Pa), Pectobacterium carotovorum subsp.carotovorum (Pcc), and Dickeya spp.cause blackleg in growing plants and soft rot in potatoes (Baghee-Ravari et al., 2011;Czajkowski et al., 2011;De Boer, 2003;van der Wolf and De Boer, 2007).Formally, they were known as Erwinia carotovora subsp.atrosepticum (Eca), E. carotovora subsp.carotovora (Ecc); and Erwinia chrysanthemi (Ech) (Czajkowski et al., 2011).Pcc has a broad host range world wide and infects potato plants in warmer summer in Europe and in hot summer in Colorado and Arizona in the USA.In contrast, Pa is restricted only to potato in temperate regions.On the other hand, Dickeya spp.infiltrates a limited number of host species in temperate, subtropical, and tropical regions (Ma et al., 2007;Toth et al., 2002Toth et al., , 2011;;Czajkowski et al, 2011).Recently, Pcc and Pectobacterium wasabie are identified as the major soft rots of storage potato in Iran (Baghee-Ravari et al., 2011).
Although potato is the third crop plant in Bangladesh after rice and wheat, little is known about the characteristics of soft rot bacterial strains.Infact, research reports on soft rot bacteria of potato are very scanty in Bangladesh (Rasul et al., 1999;Islam, 2004).Identification of causative bacterial strains help significantly take appropriate control measures, develop detection systems and resistant plants against causative strains through conventional breeding as well as genetic modifications schemes (Czajkowski et al., 2011).It also contri-butes to the understanding of their population distribution and biodiversity profiles.Considering the above facts, the present study was conducted for the biochemical identification and pathogenecity profiling of the soft rot bacterial strains in Bangladeshi potatoes.
Traditionally, the characterization and identification of pectolytic erwinias are based on biochemical and phenotypic characteristics (De Boer and Kelman, 2000) and recently molecular techniques are also applied (Baghee-Ravari et al., 2011).The most commonly used methods are biochemical tests (Dickey and Kelman 1988) and pathogenicity profiling (Smith and Bartz, 1990).Using this approach, 87 strains of the soft rot, E. carotovora subsp.carotovora (Ecc), are profiled from various host plants in Japan, South Korea, and Thailand (Seo et al., 2001).Due to the lack of molecular analytical facilities in Bangladesh, this study also adopted biochemical and physiological testing methods to identify soft rot bacterial pathogens in Bangladeshi potatoes.

Sample collection
Diseased potato plants were selected based on visible or phenotypic symptoms of soft rot and characteristic odor described by Agrios (1978), Rich (1983), andShing (2001).The soft rotted samples of potato tubers were collected from farmers' houses, different experimental farms of Bangladesh Agricultural Development Corporation (BADC), local markets, and cold storages located in different parts of Bangladesh.The selected potato varieties were Felsina, Granola, Diamant, Cardinal, Asterix, and Shilbilati and were collected from major potato farming areas of Bangladesh known as Gazipur, Baradi, Rangpur, Lalmaonirhat, Kushtia, Domar (Nilphameri), Dinajpur, Thakurgaon, Kauranbazar (Dhaka), Kashimpur, Munshiganj, and Dhaka during the cropping seasons of 2008 to 2009 and 2009 to 2010.The soft rot incidence of potato was identified according to Agrios (1997), Sherf and Macnab (1986).Briefly, the soft rot symptoms begin with small water soaked lesions, which gradually become soft, mushy, disintegrated, depressed, and discolored.The tissues within the affected region become creamy and slimy in color and gradually become disintegrated into a mushy mass of disorganized cells.Sometimes the whole tuber turns into a soft, watery, decayed mass within 3 to 5 days (Agrios, 1997).The collected samples were brought to the microbiology laboratory of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) and soft rotted bacterial strains were extracted within 24 to 48 h.

Isolation of soft rot bacterial strains
The bacterial strains were isolated from different samples of potato by "Streak plate" technique, described by Mortensen (1997) and Kim et al. (2002).A common bacteriological medium, Yeast Peptone Dextrose Agar (YPDA), was used for the extraction schemes.YPDA media was prepared by dissolving 3.0 g of yeast, 0.6 g of peptone, 3.0 g of dextrose, and 15 g of agar in 1000 ml of sterile distilled water.The pH of the medium was adjusted to 7.0 using 0. 1 M KOH and cooked on hot plate at boiling temperature until all ingredients were completely dissolved.After cooking, the medium was autoclaved for 20 min at 121°C under 1.1 kg/cm 2 pressures.The medium was poured into petridishes at the rate of 20 ml/plate and were cooled in a clean bench.To isolate the causative bacterial strains, a small part from the margin of rotted tissues of the infected tubers of potatoes were cut away with a scalpel and were surface disinfected with 1% sodium hypochlorite (NaOCl) for 2 to 3 min.Sterilized samples were washed several times with sterile distilled water to remove the residual hypochlorite.The samples were placed in petridishes containing sterile water and were crushed with a sterile scalpel.After crushing, the petridishes were kept undisturbed for 10 to 15 min to release the bacteria associated with rotted tissues.One loop full of resulting suspension (liquid containing bacteria) was streaked on the solidified YPDA medium in each plate.The plates were incubated at 30°C for 48 h.Characteristic individual bacterial colonies that appeared on YPDA medium were picked up using a bacterial loop and transferred to another plate.Purification of bacterial colony was done by restreaking of single colony on another fresh plate.

Potato soft rot test
All of the bacterial isolates originated from single colonies were tested for their ability to cause soft rot on potato tubers following procedure of Lelliot et al. (1966).Briefly, potato tubers were sterilized with 70% ethyl alcohol, rinsed with sterile distilled water, and aseptically cut into slices (ca. 1 cm).The potato slices were put in petridishes containing sterilized filter paper impregnated with ca. 2 ml of sterile distilled water.The soft rot tests were repeated at least thrice.The potato slices were inoculated with needle pricking method.The inoculated slices were maintained in moistened petridishes according to Togashi (1988) and Nabhan et al. (2006) and incubated at 30°C for 2 to 3 days.The bacterial cultures that produced characteristic symptoms of soft rot on potato slices were selected and preserved in test tubes containing YPDA media overlayed with sterile liquid paraffin at 4°C in a refrigerator for further studies.
Three soft rotting bacterial strains, E. carotovora subsp.carotovora (Ecc) ATCC-15713, E. chrysanthemi (Ech) Ura-2, and Burkholderia cepacia (Bca) ATCC 25416 were obtained from the microbiology laboratory BSMRAU, previously collected from the Kyushu University, Japan and were used as reference strains in this study.

Collection and Isolation of soft rot bacterial isolates
A total of 92 bacterial strains from potato were isolated from different locations of Bangladesh (not shown).Colony morphologies of most of the isolates on YPDA media were white, creamy white, and grayish creamy white, smooth, round, glistening, slightly raised, and some were flat to slightly raised, margins undulated to feathery, and visible on isolation plates after about 24 h (Figures 1a and b).

Potato soft rot test
Based on the results of potato soft rot test, the pathogenic isolates were selected from the extracted 92 potato isolates.Out of the 92 bacterial isolates, 26 isolates (Table 1) produced soft rot on potato slices (Figures 2a and b).All of the pathogenic isolates were preserved in test tubes containing YPDA media, overlayed with liquid paraffin for further study.Out of the 26 soft rot-positive isolates, 12 were found aggressively virulent and were selected for further characterization and identification.
All the 12 virulent isolates and reference strains except Bca ATCC 25416 were OF positive (Figure 4) and developed characteristics red color of glucose reduction.Among the 12 isolates of potato, two isolates, designated as P-128 and P-179 and the reference strain of Ech Ura-2, were positive in the lecithinase test while the rest of the 8 isolates, designated as P-01, P-04, P-11, P-48, P-111, P-134, P-138, and P-169 as well as the reference strains of Ecc, ATCC 15713, and Bca ATCC 25416 were negative in lecithinase test.On the contrary, two isolates, designated as P-28, P-142 showed variable reaction in the lecithinase test (Table 2).
In case of methyl red test, 8 isolates marked as P-01, P-04, P-11, P-48, P-111, P-128, P-134, P-138, and P-169 and the reference strain of Ecc ATCC 15713 developed red color, demonstrating positive reaction, whereas the rest of the isolates marked as P-28, P-128, P-142, and P-179 and the reference strain of Ech Ura-2 retained the original color of the solution, showing the negative reaction (Table 2 and Figure 7).
The isolates P-28, P-128, P-142, and P-179 and the reference strain of E. chrysanthemi Ura-2 were positive in the catase test showing the formation of characteristic bubble (Figure 3).The rest of the 8 strains marked as P-01, P-04, P-11, P-48, P-111, P-134, P-138, and P-169 and the reference strain of Ecc ATCC 15713 did not produce any gas bubbles in this particular test (Table 2 and Figure 3).

The Carbon source utilization test
All of the isolates of potato utilized cellubiose, Larabinose, D-galactose, D-xylose, raffinose, sucrose, and manitol, as sole sources of carbon but did not use maltose, dulcitol, sorbitol, benzoate, and D-tartrate for their growth (Tables 3 and 4).However, some of the isolates differed in in the type of carbon source utilization.

DISCUSSION
Bacterial soft rot is one of the most devastating diseases of Bangladeshi potatoes.The disease is characterized by foul-smelling rot and collapse of the potato tubers.In the present study, a serious degree of soft rot occurance was found in Bangladeshi potatoes in storage conditions.Among the 12 selected virulent strains, 8 isolates were identified as E. carotovora subsp.carotovora (Ecc) and 4 isolates were identified as E. chrysanthemi (Ech).These findings strongly correlated with the typing results of Kreig and Holt (1984), Karnjanarat et al. (1987), Alam et al. (1999), andKhan et al. (2000) for Ecc and Ech.Usually, Eca causes soft rot and blackleg in potatoes in cool and dry temperate climates.On the other hand, Ecc and Ech are the causative agents of blackleg and soft rot in the temperate regions (<25°C) (Pérombelon and Kelman, 1980).Since the Bangladeshi climate is moderately temperate and humid, the findings of Ecc and Ech as major soft rot bacterial pathogen were logically sound.To characterize the pathogenic strains, this study used biochemical and physiological techniques as the characterization tools.Since the molecular characterization tools are more reliable than those of the biochemical and physiological ones (Baghee-Ravari et al., 2011), the results of the present study should be validated

Figure 1 .
Figure 1.Various types of bacterial colonies on YPDA media isolated from soft rotted potato.Shown are: (a) white, smooth, and flat to slightly raised colonies, and (b) white round colonies on YPDA media.

Figure 2 .Figure 3 .
Figure 2. Soft rot test of two bacterial strains, P-01 (a), and P-128 (b) on potato slices.The positive (left) and negative (right) results are shown side by side.

Fig. 3 Fig. 4
Fig.3Catalase test of soft rot bacteria.An example of positive test is shown in (A) with trapped gas bubles, negative test is shown in (B) with no detectable gas bubles.

Figure 4 .Fig. 5 BFigure 5 .
Figure 4. Oxidative and fermentative tests.Shown are: positive reaction (A and B) negative reaction (C and D) and negative control (without glucose added) (E).a.An example of positive test is shown in (A) with st is shown in (B) with no detectable gas bubles.

Rahman et al. 1441 Fig. 4
Fig. 4 Oxidative and fermentative tests.Shown are: positive reaction (A and B) negative reaction (C and D) and negative control (without glucose added) (E).

Figure 6 .
Figure 6.Tobacco hypersensitivity (HR) test: positive test is shown in (A) with a significant portion of tissue collapsed and necrosis within 24 h of inoculation and the negative test is shown in (B) where no sign of tissue collapsed and necrosis was observed under similar treatment.

Fig. 3 Fig. 4
Fig. 3 Catalase test of soft rot bacteria.An example of trapped gas bubles, negative test is shown in (B)

Table 1 .
The soft rot positive bacterial isolates of potato tubers collected from different parts of Bangladesh.

Table 2 .
Physiological and biochemical characteristics of soft rot bacterial isolates of Bangladeshi potatoes.

Table 5 .
Identified soft rot bacterial pathogens isolated from potato tubers collected from 10 different potato farming areas of Bangladesh.Bangladesh and the publication cost was realized from Malaysian Ministry of Science, Technology, and Innovation grant No. 05-01-35-SF-1030" to Prof. U. Hashim.