Identification and Population Estimation of Epiphytic Ice Nucleation Active Bacteria Isolated from Stone Fruit Trees in Khorasan-Razavi Province

Epiphytic bacteria are common inhabitants of plant leaflet surfaces and can have important influences on the plants on which they live. Accurate estimates of epiphytic bacterial population sizes are needed to detect their roles in several important biological phenomena. For example, reliable disease forecasts and anticipate of frost damage depend on the exact estimation of bacterial population in their habitant. Frostbite is one of the major agricultural problems in Iran and is one of the most important environmental stresses which influences plant growth and crops productivity. Chilling and freezing limit the geographical distribution and growing season of many crops and cause significant crops losses. Apical meristem and upper leaves show the first symptoms of frostbite. Without bacteria involved, freezing damage on actively growing shoots is usually limited to the tip or outer branches, and the plant will recover. Freezing is not the sole reason of severe damage to such plants and if such damage or dieback occurs there must be synergistic effects between frost and bacterial infection. Ice nucleation epiphytic bacteria are one of the casual agents of frost and canker in stone fruit trees. In order to identify ice nucleation active (INA) bacteria, 248 samples from twigs of sour cherry, sweet cherry, apricot, prune, peach, nectarine, almond and plum from the main areas of stone fruit tree orchards in Khorasan-Razavi province were collected in spring of 2013. In each area, 2 orchards and in each orchard 4 trees were randomly selected. The samples were placed in paper bags and were kept in a refrigerator until use. The weight of tissues was measured and bacteria were washed from the surface of plant parts in distilled water treated with 0.2 percent. The samples were homogenized for 1 hour at 100 rpm rotary shaker. One milliliter of this suspension was serially diluted to 10−9, 10−8, 10−7 and 10−6 cfu/ml. Twenty-five micro liter of each dilution was cultured on King’s medium B. Among bacterial colonies grown after 48 h, 820 isolates were randomly selected (10 colony of each types that observed on petri dishes). Bacterial population in different areas and hosts, were estimated by counting of the bacterial colonies in each gram of fresh weight. To discover INA bacteria, bacterial suspensions in distilled water were incubated at -5 to -7 °C. After 30 min freezed bacterial suspensions were selected. The population of INA bacteria were estimated by the formula -. The ice nucleation active bacteria were purified and identified by different tests including Gram reaction, catalase, fluorescent pigment production on KB or casamino acid medium, levan, oxidase, arginine dihydrolase, potato rot and hypersensitive reaction on tobacco. The growth with 5% and 7% NaCl, anaerobic condition, at 37 °C, were also tested. The yellow bacteria were cultured on YDC, and the production of xanthomonadin was tested. Among these 820 isolates that analyzed, 110 isolates were determined as INA. These bacteria were identified by phenotypic and biochemical tests. According to our data 56, 27, 8, 6 and 3 percent isolates were identified as Pseudomonas syringae, P. fluorescens, P. viridiflava, Pantoea agglomerans and Xanthomonas sp., respectively. The population of INA bacteria in chenaran and Mashhad was higher than other cities. There was no correlation between plant species and the population of INA bacteria. Although our data indicated the positive correlation between canker symptoms and the population of INA bacteria. This study indicated that plant species cause significant differences in bacterial population size. Effects of plant species on epiphytic bacterial population sizes may also reflect physiological differences. It is fascinating that, for some species, different plants of the same genotype grown in the same area and under similar environmental conditions would exhibit significant differences in mean bacterial population size. It is possible that diversity in soil and water content, soil nutrient availability, release nutrient deposition, nutrient leakage from plants, presence of unseen wounds through which leakage occurs, or exposure to immigrants contribute to the generation of significant differences in bacterial population sizes among plants. Perhaps bacterial population sizes on plant species in which resources required for epiphytic colonization are abundant and are less susceptible to influenced by exogenous factors, therefore fewer variables among plants. We found that bacterial population sizes were varied in relation to plant host, and the physical environment. The ice formation and frost damage in plants are normally depend on the logarithm of the ice nuclei number in freezing time. Therefore, population estimation of the epiphytic INA microorganisms is very important for frost forecasting. Our results, for the first time, demonstrated the population dynamics and the presence of different species of ice nucleation active bacteria in Khorasan-Razavi province.