Bacterial crown and root rot disease of alfalfa is caused by a Gram negative and fluorescent bacterium Pseudomonas viridiflava [1]. This is one of the important diseases of alfalfa, which causes crop quantity and quality losses. The bacterium was first recognized by Burkholder, then named as Phythomonas viridiflava by Dowson, and finally changed to P. viridiflava [2]. P. viridiflava damages alfalfa plants at all growth stages. Infected seedling show symptoms which includes wilting, chlorosis, stunting, leaf yellowing and plant malformation. Plants in developed disease stage show dwarfing, and growth causal bacterium causes wound and rotting on plant roots and changes the root color. Wilting symptoms are very obvious, especially under warm condition. The bacterium is more active under humid and raining condition, and it can spread through the rain drops [3]. P. viridiflava has broad host range and causes leaf blight on melon, tomato, pumpkin, cauliflower, cabbage, grapevine, lettuce, carrot, peas, poppy, turnip and eggplant, wilt of sweet onion, and canker and nude blight of kiwi [4-6]. This bacterium was observed as second pathogen for pith necrosis of tomato and pepper, and has ability of transfer through the seeds in tomato and pepper. P. viridiflava is an agent of dwarfing in Fabacea plants and causes leaf necrosis, dwarfing and root rot of Trifolium pretense [1]. Also it assist P. corrugate to cause pith rot on several plants [2], and increase disease symptoms arising from soil borne nematodes such as Meloidogyne hapla, pratylenchus penetrans, Ditylenchus dipsaci and Helicotylenchus dihystera [7].

Using specific primers for detection and recognition of bacterial strains is very serviceable, and each organism could be recognized directly with PCR-based studies, in least time [8]. DNA-based methods, such as PCR have provided accurate description of genetic variation and phylogenetic relationships, among the pathogens. This technique is more sensitive than cultural or serological methods, and is rapid and specific for plant pathogens [9]. Alfalfa is one of the most important forage crops in Hamedan province of Iran. Climatic condition in this province is suitable for alfalfa growth but many diseases, including alfalfa bacterial crown and root rot disease, cause economic losses of this crop. As this disease is new, the main aim of this study was characterization of the causal agent of the disease in Hamedan province, by classical and molecular methods.

Bacterial isolation

During 2009 spring, wilted and suspicious alfalfa plants were collected from the main growing areas in Hamedan province, located west of Iran. Plant roots samples were disinfected in 70% ethanol, following by 0.5% sodium hypochlorite for 5 minutes, washed twice in sterilized distilled water, grinded in phosphate buffer and shake (100 rpm) for 1 hr at room temperature [6]. A loop-full of bacterial suspension was stroked on the plates, containing nutrient agar medium enriched with yeast extract and glycerol, and they were kept at 25°C for 2-3 days. To purify the bacterial isolates, olive to golden brown color single colonies were selected and re-stroked on plates containing the above medium. Selected bacterial isolates were kept, either in sterilized distilled water [10] or on medium slants in test tubes, under sterilized liquid paraffin for further investigations.

Pathogenicity test

To prepare the plant seedlings for pathogenicity test, healthy alfalfa seeds were disinfected in 5% sodium hypochlorite for 10 minutes, and they were washed three times in sterilized distilled water. Fifteen seeds were planted in each pots containing two kilogram sterilized loamsandy soil, under greenhouse condition in three replicates. Bacterial strains were grown in nutrient yeast extract broth (NBY) and their pellet was obtained by centrifugation. Suspensions of bacterial strains with optical density 0.1 (OD=0.1) at 600 nm wavelength were prepared using spectrophotometer, and they were injected into the stem of alfalfa seedlings (3 to 4 leaves stage). At the same time, bacterial suspensions with the same concentration were added to the soil [1]. Sterilized distilled water was injected or added to the soil, as a control treatment. A total of 35 bacterial isolates which showed apparent characteristics of suspected causal agent of the alfalfa root rot disease, were used in pathogenicity test.

Phenotypic characterization of the bacterial strains

As bacterial isolates were uniform and showed similar pattern of pathogenicity, 15 representatives together with two defined P. viridiflava strains gifted by collection of laboratory of bacteriology, Iranian Plant Disease Research branch, were used for phenotypic feature characterization. Following physiological characteristics were determined, based on the standard bacteriological methods: Gram reaction using 3% KOH [11], Gram staining [10], hypersensitivity reaction on Tobacco, Geranium, Petunia and Tomato plants [12], oxidase [12] and oxidative/fermentative (O/F) test [13]. Also, gelatin liquefaction, levan formation, acid from trehalose, sucrose, Darabinose, cellobiose, sorbitol, glucose, fructose and manitol, urease, growth at 37°C, fluorescent pigment on King’s B medium, arginine dihydrolase, and pectinolytic activity on potato slice were performed [5,10,14].

PCR assay

DNA from bacterial isolates was extracted according to Ausubel et al. [15] with minor modifications. In brief, bacterial strains were grown in NBY medium for three days, and their cell pellet were precipitated by centrifugation. Bacterial pellets re-suspended in 100 μ TE buffer and boiled for 5 minutes, for DNA releasing. After cooling, bacterial debris was precipitated by centrifugation (10000 rpm), and 500 μl from supernatant of each aliquot were kept at -20°C.

Amplification of target sequences of the bacterial DNA was performed using primers: Ten isolated bacteria together with two defined P. viridiflava strains gifted by collection of laboratory of bacteriology, Iranian Plant Disease Research branch, were used in primary PCR experiment. This experiment was conducted using a set of primers (PVF/ PVR) designed for 16S rDNA sequence of P. viridiflava LMG 2352T (type strain), obtained from NCBI database with accession number Z76671. The primer sequence of PVF was 5’-TCAACCTGGGAACTGCATCC- 3’ and PVR was 5’-ATCACACCGTGGTAACCGTC-3’. PCR experiment was conducted at 58°C annealing temperature, for 35 cycles. Then, two sets of primers were used for amplification of target sequences, only in 10 isolated bacteria from alfalfa plants in Hamedan province (Iran). Sequences of these primers sets were 7F (5’-GTTCCTTGAAGTGCCTGA- 3’) / 7R (5’-GTTTTCGTAGCGGTTGCG-3’) and 20F (5’-CGCCGTTTTCGTTCTTGT-3’)/ 20R (5’-GCATGGAATACGCCGACA- 3’). For these two sets of primers, PCR experiments were conducted at 50°C and 58°C annealing temperature, respectively, for 35 cycles. PCR reaction contained 1 μl of primers 0.2 μM, 2.5 μl buffer 1X, 2 μl MgCl2 2.5 mM, 0.5 μl dNTPs 0.2 mM, 0.5 μl Taq DNA polymerase, 1 μl DMSO 2.5 mM, and 1 μl of target DNA solution, in 25 μl final reaction volume.

Bacterial isolation

A total of 35 typical bacterial isolates were selected from bulk isolates, sampled from alfalfa growing area of Hamedan province. In 25°C, the olive to golden brown color colonies of isolated strains appeared after 3 days (Figure 1).

Figure 1: Colonies of Pseudomonas viridiflava isolated from alfalfa plants in Hamedan province (Iran) on nutrient agar medium.

Pathogenicity test

After 40 days, infested alfalfa plants showed symptoms of the disease, including stunting and wilting. All of 35 bacterial isolates induced disease on alfalfa seedlings in green-house, and they showed similar pattern of pathogenicity, in both methods of inoculation, which includes plant stem injection and soil drenching. Under green house condition, disease symptoms were developed rapidly and severely, especially when bacterial suspension was injected into plant stem in comparison to soil drenching. In later method, disease symptoms appeared after 65 days (Figure 2).

Figure 2: Pathogenicity of Pseudomonas viridiflava on alfalfa plants. A: greenhouse experiment; B; infected plants and C: healthy plants.

Phenotypic characteristics of the bacterial strains

As 35 isolated strains showed identical features of colony color and shape, growth and pathogenicity pattern, 15 strains were randomly selected for phenotypic feature characterization. These strains together with two defined P. viridiflava strains gifted by collection of laboratory of bacteriology, Iranian Plant Disease Research branch were characterized phenotypically. Gram reaction tests showed that all strains were Gram negative (Table 1).

Table 1: Hypersensitive reaction of Pseudomonas viridiflava strains isolated from alfalfa plants in Hamedan province (Iran) on some plants.

All tested strains induced hypersensitive reaction (HR) on Tobacco, Geranium and Tomato leaves (Table 2). Based on the results of phenotypic feature determination, all tested bacterial strains were identified as P. viridiflava (Table 3). This new reported bacterium is less pathogenic than Clavibacter michiganensis subsp. insidiosus on alfalfa, in Iran. This disease and its causal agent previously has never been reported from other alfalfa growing area in Iran, even as an alfalfa plants associated bacteria.

Table 2: Some phenotypic characteristics of 15 representatives and defined Pseudomonas viridiflava strains isolated from alfalfa plants in Hamedan province (Iran).

Table 3: Identification of the 15 representative strains causing alfalfa root rot disease in Hamedan Province (Iran).

PCR results

A set of primers PVF/PVR yielded a 860 pb PCR band in all tested bacterial isolates, including defined P. viridiflava strains as expected from 16S rDNA sequence of P. viridiflava LMG 2352T (Figure 3). This criterion confirms the causal agent of alfalfa root rot, to be P. viridiflava. The primers sets 7F/7R amplified nearly equal to 400 bp bands (Figure 4) and primers set 20F/ 20R amplified nearly equal to 440 bp band in all tested strains (Figure 5).

Figure 3: An 860 bp PCR amplified fragment of Pseudomonas viridiflava strains using PVF1/PVR1primers designed for 16S rRNA sequence. From the right side: First lane DNA size marker; second and third lanes a defined P. viridiflava strains and other lanes AH4, AH8, AH11, AH16, AH18, AH23 & AH25 strains isolated from alfalfa plants in Hamedan province(Iran).

Figure 4: PCR amplified fragment of Pseudomonas viridiflava strains isolated from alfalfa plants in Hamedan province (Iran) using 7F and 7R primers.

Figure 5: PCR amplified fragments of Pseudomonas viridiflava strains isolated from alfalfa plants in Hamedan province (Iran) using 20F and 20R primers.

P. viridiflava which causes alfalfa crown and root rot disease also affects melon, tomato, pumpkin, cauliflower, cabbage, grapevine, lettuce, carrot, peas, poppy, turnip, eggplant, sweet onion, kiwi, and clover [4,6,16]. The causal bacterium of the disease is found in the intercellular spaces of the seed parenchyma [17]. Bacterial crown and root rot disease is one of the important diseases of alfalfa, and causes economic losses in Hamedan province of Iran, where until now, no study was performed on the disease and its causal agent. All tested bacterial isolates in this study induced typical disease symptoms, caused by P. viridiflava on alfalfa seedling (Figure 2). The disease symptoms during sampling in Hamedan alfalfa growing area were same, as reported by Lukezic et al. [1] and Jones et al. [3], which include chlorosis, stunting and wilting. However severe stunted plants were observed rarely in diseased plants and this symptom was often observed in plants with high developed symptoms. This may indicate the importance of physical obstruction of vessels by bacterium, and inhibition of nutrient material transfer to upper parts of the plant. Some researchers showed observable symptoms developed by P. viridiflava on alfalfa, after 50 days after adding bacterial suspension into the soil, and after 30-40 days when they were injected into plants [1,3]. In our experiments, disease symptoms were observed after 40 days when bacterial suspension injected into the plants stem, and after 60 days when soil was infested by bacterial suspension. This result is in accordance with those reported by Lukezic et al. [1] and Jones et al. [3].

Hypersensitive reaction (HR) on tobacco leaves were observed as previously reported by other researchers [3,6]. However, this is the first report of HR on leaves of Geranium and Tomato by P. viridiflava. Results of phenotypic feature characterization, including LOPAT tests for bacteria isolated from diseased alfalfa plants, and two defined P. viridiflava strains were same, as described by others reports [10,14].

The PCR-based identification methods for P. viridiflava are very rapid, sensitive and specific. In our study, the use of specific primers had lead to verification of the results of physiological tests, and the certainty of the presence of P. viridiflava in Hamedan province. An 860 pb expected PCR band in all tested bacterial isolates, including defined P. viridiflava strains using PVF/PVR primers set, confirms the causal agent of alfalfa root rot to be from P. viridiflava. This primer set was designed for amplification of 16S rDNA sequence, based on nucleotide database of P. viridiflava LMG 2352T derived from NCBI website. Also the 7F/7R and 20F/ 20R primer sets amplified nearly equal to 400 and 440bp, respectively. These observations in our PCR experiments were same to those reported by Goss et al. [18]. It is worthy to note that as this is the new reported bacterium causing alfalfa crown and root rot in Hamedan province, and there is no report on other alfalfa growing area, there is a possibility that the causal agent might be present in other area and caused crop losses. Therefore, survey for the presence of the bacterium causing alfalfa crown and root rot disease is very important and necessary, for disease control and management in future studies.