Ice Nucleation Active Bacteria Research Articles

A sustainable method to increase the strength of warm permafrost: Ice nucleation active bacteria-based

Ice nucleation active (INA) bacteria are capable of triggering ice formation close to 0 °C, but their ability of increasing ice content in warm permafrost remain unknown. Ice content is vital because it determines the bearing capacity of warm permafrost. Through nuclear magnet resonance and direct shear device, we found that adding INA bacterium Pseudomonas syringae with a concentration of 1 g/L in warm frozen soil can result in 64% increase in the shear strength, 113% increase in cohesion and 27% increase in ice content. The internal friction angle of warm frozen soil is less affected by P. syringae. Warm frozen soil with P. syringae exhibits brittle failure under normal stresses of 100 kPa to 300 kPa and plastic failure under 400 kPa. The shear strength increment can be regulated by the concentration of P. syringae which exponentially relates to ice content and linearly to shear strength. This emerging strategy reveals the importance of INA bacteria in cooling permafrost, and provides a sustainable and environment-friendly method for confronting permafrost degradation and the subsequent infrastructure instability.

Ice Nucleation Active Bacteria in Vineyards and Orchards and Low Temperature Damage

Ice Nucleation Active Bacteria in Vineyards and Orchards and Low Temperature Damage

The Effect of Ice-Nucleation-Active Bacteria on Metabolic Regulation in Evergestis extimalis (Scopoli) (Lepidoptera: Pyralidae) Overwintering Larvae on the Qinghai-Tibet Plateau.

Simple SummaryEvergestis extimalis (Scopoli) causes a serious reduction in the yield of spring oilseed rape every year. Until now, using chemosynthesis pesticides has been the main strategy to control E. extimalis. Biopesticide, as an alternative treatment, has attracted widely attention as it has little effect on the natural enemy and a low potential for promoting resistance. Our results have indicated that ice-nucleation-active microbes were able to stimulate trehalase activity, thereby leading to the reduction of trehalose accumulation. Consequently, E. extimalis showed an obviously decreased survival rate and ability to withstand low temperatures in the Qinghai-Tibet Plateau. Therefore, the use of INA bacteria has the potential to become a new strategy for the biological control of pests.Evergestis extimalis (Scopoli) is a significant pest of spring oilseed rape in the Qinghai-Tibet Plateau. It has developed resistance to many commonly used insecticides. Therefore, biopesticides should be used to replace the chemical pesticides in pest control. In this study, the effects of ice-nucleation-active (INA) microbes (Pseudomonas syringae 1.7277, P. syringae 1.3200, and Erwinia pyrifoliae 1.3333) on E. extimalis were evaluated. The supercooling points (SCP) were markedly increased due to the INA bacteria application when they were compared to those of the untreated samples. Specifically, the SCP of E. extimalis after its exposure to a high concentration of INA bacteria in February were −10.72 °C, −13.73 °C, and −14.04 °C. Our findings have demonstrated that the trehalase (Tre) genes were up-regulated by the application of the INA bacteria, thereby resulting in an increased trehalase activity. Overall, the INA bacteria could act as effective heterogeneous ice nuclei which could lower the hardiness of E. extimalis to the cold and then freeze them to death in an extremely cold winter. Therefore, the control of insect pests with INA bacteria goes without doubt, in theory.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Ice nucleation active bacteria and its mitigation on tomato (Solanum lycopersicum)

Microbes play a vital role in ice nucleation, supporting bio-precipitation, and allowing plants to live in low-water environments. A field experiment was conducted during December 2018 with two phyllosphere microorganisms’ spraying viz., Pseudomonas aeruginosa and pink-pigmented facultative methylotrophs (PPFM) and under three moisture regimes (0.6, 0.8, 1.0 IW/CPE (irrigation water/ cumulative pan evaporation) ratio) on tomato (PKM 1) in Tamil Nadu Agricultural University, Coimbatore (Tamil Nadu). A laboratory experiment was conducted to confirm ice nucleation using two phyllosphere microorganisms’ P. aeruginosa and PPFM. The bioprecipitation impacts on tomatoes were assessed using a set of physiological parameters such as photosynthetic rate, average chlorophyll Index and the ice nucleation activity (INA) assessed using tube nucleation test and scanning electron microscope (SEM). The results showed that the mean photosynthetic rate of PPFM sprayed tomato (Solanum lycopersicum) (PKM 1) plants (40.7 μmol CO2/m2/s) at 10 DAS was significantly higher than P. aeruginosa sprayed plant (38.7 μmol CO2/m2/s) under different irrigation regimes. The average chlorophyll Index value of the P. aeruginosa sprayed tomato plants (58.1) was higher than PPFM sprayed plants (56.4). The tube nucleation tests were proved that ice crystallization induced by P. aeruginosa in super-cooled buffer at - 2 to -10°C while PPFM not catalyze the buffer even after 3hours. The scanning electron microscope (SEM) indicated the P. aeruginosa growth at the upper surface of the leaf and PPFM growth more at the lower surface of the leaf compared to without inoculation of microbes on leaves. Overall, the result revealed that P. aeruginosa may assist in ice nucleation activity that will help to make artificial rain in the near future.

Ice Nucleation Active bacteria in Mount Lawu forest, Indonesia: 2. Identification and characterization of ina gene bacteria isolated from lichens

Abstract. Arifin TN, Susilowati A, Sutarno. 2018. Ice Nucleation Active bacteria in Mount Lawu forest, Indonesia: 2. Identification and characterization of ina gene bacteria isolated from lichens. Asian J For 2: 39-46. Ice nucleation active (INA) bacteria can catalyze ice formation. Moreover, these bacteria cause frost injury in plants. This study aimed to determine the ina bacteria species of Mount Lawu forest, Java, Indonesia, based on the 16S rRNA gene and the characters of ice nucleation active gene coding of ina bacteria on lichens used ina gene primer. First, the isolates of ina bacteria were grown in NAG, and the DNA could be isolated. After that, genes coding for 16S rRNA and ina were amplified, and the amplification products were sequenced. Furthermore, the sequences were analyzed with BLAST program to know the similarity of bacteria species and ina gene characters. The result showed that the isolated ina bacteria had similarities with Pantoea, Pseudomonas, and Rahnella. The ina gene from N.2.1.B-13 isolate consists of 189 amino acids, dominated by a common amino acid found in other ina genes, like Alanine, Glycine, Tyrosine, Serine, Leucine, and Threonine. N terminal and C terminal are not found because ina gene from N.2.1.B-13 isolate is similar to the ice nucleation protein gene Pseudomonas borealis from bases 2947 bp to 3491 bp or 871 to 1060 amino acids.

Ice Nucleation Active bacteria in Mount Lawu forest, Indonesia: 1. Isolation and estimation of bacterial populations on lichens

Abstract. Fu’adah K, Sari SL, Susilowati A. 2017. Ice Nucleation Active bacteria in Mount Lawu forest, Indonesia: 1. Isolation and estimation of bacterial populations on lichens. Asian J For 1: 83-91. Ice Nucleation Active (INA) bacterias cause frost injury in plants. These bacteria are also predicted to play an important role in bioprecipitation, cloud formation, and rain. Most INA bacteria studies are conducted in subtropical areas. Therefore, studies on INA bacteria from tropical areas, especially Indonesia, need to be conducted. This study aimed to isolate and determine the number of INA bacteria in lichenes. samples of lichenes were taken from the hiking pathway of Cemoro Sewu, Mount Lawu forest, Java, Indonesia, at ±2.200, ±2.400, and ±2.600 m asl. INA bacteria were isolated by the spread plate method on the NA medium with 2.5 % glycerol and King’s B medium. A tube nucleation test determined ice nucleation activity. A multiple-tube nucleation test estimated the number of INA bacteria. The data were analyzed descriptively based on colony morphology, cell morphology, biochemical tests, and analysis of the numbers of INA bacteria. The result showed that 7 isolates from Parmelia sp. at an altitude of 2.532 m asl (station 2) have been known as INA bacterias. The number of INA bacteria in lichenes was 5 x 104/g, which was very low.

Prevalence and characterization of Ice Nucleation Active (INA) bacteria from rainwater in Indonesia

BackgroundIce nucleation active (INA) bacteria are a group of microorganisms that can act as biological nucleator due to their ice nucleation protein property. Unfortunately, little is known about their prevalence and characteristics in tropical areas including Indonesia. Here, we monitor the presence of INA bacteria in rainwater and air samples collected from Jakarta, Tangerang and several areas in Western Java, Indonesia for one year. We further identify and characterize selected Class A of INA bacteria isolated from these areas.ResultsMost of the INA bacteria were isolated from rainwater samples collected during March–August 2010, particularly from Jakarta, Bandung, and Tangerang. A total of 1,902 bacterial isolates were recovered from these area. We found a limited number of bacterial isolates from air sampling. From ice nucleation activity assays, 101 INA isolates were found active as ice nucleator at a temperature above -10 °C. A large majority (73 isolates) of them are classified as Class C (active below -8 °C), followed by Class A (26 isolates; active at -2 to -5 °C) and Class B (two isolates; active at -5 to -8 °C). We sequenced the 16S rRNA gene of 18 Class A INA isolates and identified 15 isolates as Enterobacteriaceae, while the remaining three as Pseudomonadaceae. The vast majority of our Class A INA isolates were likely Pantoea spp. with several isolates were deduced as either Pseudomonas, Cronobacter, and Klebsiella. We found that these 18 Class A INA isolates had acquired resistance to antibiotics erythromycin and ampicillin, which are considered two critically important antibiotics.ConclusionsOur results showed that the prevalence of INA bacterial population varies across locations and seasons. Furthermore, our isolates were dominated by Class A and C INA bacteria. This study also cautions regarding the spread of antibiotic resistance among INA bacteria.

Biological Ice Nucleators in Snow Samples from Greece

The formation of precipitation in clouds is initiated by inorganic and organic/biological ice nuclei. Certain species of bacteria and fungi are known to act as efficient biological ice nuclei at temperatures between −10 and 0 °C. Biological ice nuclei have been found and characterized in precipitation samples (snow, rain, and hail). We investigated the presence of warm temperature biological ice nuclei in 17 fresh snow samples from Greece and isolated and partially characterized ice nucleation active bacteria from these. All snow samples contained particles or other material active as ice nuclei at −9 °C in concentrations ranging from 3 to 943 nuclei/L. The numbers of this class of ice nuclei were reduced or eliminated after incubating snowmelt concentrates at 100 °C for 15 min and by treatment with lysozyme, a bacterial cell wall-degrading enzyme. These findings indicate the presence of microbial ice nuclei in snow samples from Greece. We also isolated ice nucleation active bacteria from some of the samples. These bacteria belong to genus Pseudomonas and are common on plants and soil. This is the first report on biological ice nuclei in precipitation samples from Greece.

Ice-nucleating proteins are activated by low temperatures to control the structure of interfacial water

Ice-nucleation active (INA) bacteria can promote the growth of ice more effectively than any other known material. Using specialized ice-nucleating proteins (INPs), they obtain nutrients from plants by inducing frost damage and, when airborne in the atmosphere, they drive ice nucleation within clouds, which may affect global precipitation patterns. Despite their evident environmental importance, the molecular mechanisms behind INP-induced freezing have remained largely elusive. We investigate the structural basis for the interactions between water and the ice-nucleating protein InaZ from the INA bacterium Pseudomonas syringae. Using vibrational sum-frequency generation (SFG) and two-dimensional infrared spectroscopy, we demonstrate that the ice-active repeats of InaZ adopt a β-helical structure in solution and at water surfaces. In this configuration, interaction between INPs and water molecules imposes structural ordering on the adjacent water network. The observed order of water increases as the interface is cooled to temperatures close to the melting point of water. Experimental SFG data combined with molecular-dynamics simulations and spectral calculations show that InaZ reorients at lower temperatures. This reorientation can enhance water interactions, and thereby the effectiveness of ice nucleation.

JENIS DAN POPULASI BAKTERI ICE NUCLEATION ACTIVE PENYEBAB LUKA BEKU PADA DAUN JERUK KEPROK SOE DI DATARAN TINGGI MUTIS

Research on ice nucleation-active bacteria causes frost injury from tropic areas has not been widely publicized. The purpose of this study was to determine the population of Ice Nucleation-Active Bacteria on Soe tangerines leaves and the class of Ice Nucleation-Active bacteria based on Ice formation temperatures. The collecting of Soe tangerine leaves used the purpose sampling method. Leaves with frost blotches were collected from three stages at altitudes of 1500, 1800, and 2000 meters above sea level (m asl). Bacterial isolation was carried out by the spread plate method on Nutrien Agar 2,5 % glycerol (NAG) media. Ice Nucleation activity was determined by the tube nucleation test method. Estimation of INA bacterial population was conducted by the multiple-tube nucleation test with Thomas series .3.3.3. The result showed that the highest INA bacterial population was 6.9x104 which was found in leaves samples collected from stations 1800 and 2000 m asl, and the lowest population i.e. 5,4x103 on leaf samples from station 1500 m asl. Based on the temperature of ice formation, it was known that INA bacteria that attack the Soe tangerines leaves Mutis plateau are the INA bacteria class B and C.

Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ.

Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N- and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at -20.1 °C and with inner membrane anchored INPs at a T50 value between -7 and -9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between -6 and -8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T50 of -18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.

Microbial composition in seasonal time series of free tropospheric air and precipitation reveals community separation

Primary biological aerosols are transported over large distances, are traveling in various media such as dry air masses, clouds or fog, and eventually deposited with dry deposition, especially for larger particles, or precipitation like rain, hail or snow. To investigate relative abundance and diversity of airborne bacterial and fungal communities, samples have been collected with a liquid impinger (Coriolis µ) from the top of Mount Sonnblick (3106 m asl, Austrian Alps) from the respective sources under a temporal aspect over four seasons over the year to include all climatic conditions. Bacterial and fungal samples (16S rRNA and ITS) were sequenced using Illumina MiSeq paired-end sequencing, investigated for relative abundance by qPCR(16S rRNA and 18S rRNA) and ice nucleation activity. Results show that there is no stable free tropospheric air microbial community and air mass origin was different for the four sampling periods which exerted influence on the microbial composition of the atmosphere although a core microbiome could be identified consisting of 61 bacterial OTUs and eight fungal genera. Differentiation between seasons was stronger pronounced in air than in precipitation, with rain being most different and variable of precipitation types, indicating distinct forces driving microbial fate in the air. Microorganisms precipitated with snow, hail or rain or being transported by clouds differ in their species composition from free tropospheric air masses and do not mirror the air community structure. They were more diverse, distinct in composition, 16S:18S ratio and abundance from free-floating PBA. Hence, snow or cloud samples are not suitable proxies for free tropospheric air microbiome composition, since separation processes in aerosolization, transport and scavenging occur. The microbial composition of arriving precipitation or clouds represents only a part of the microbial air composition communities of the cumulative sources of origin. Relative abundance and composition of ice nucleation-active bacteria showed a higher share of relative % reads of known ice nucleation-active bacteria present in all wet phases compared to air. Results propose a separation of IN-active reads with higher shares occurring in precipitation. This study presents the first comparison of free tropospheric bacterial and fungal abundance and diversity in time series of air over several seasons in contrast to various precipitation forms in the free troposphere.

Microorganisms Collected from the Surface of Freshwater Lakes Using a Drone Water Sampling System (DOWSE)

New tools and technology are needed to study microorganisms in freshwater environments. Little is known about spatial distribution and ice nucleation activity (INA) of microorganisms in freshwater lakes. We developed a system to collect water samples from the surface of lakes using a 3D-printed sampling device tethered to a drone (DOWSE, DrOne Water Sampling SystEm). The DOWSE was used to collect surface water samples at different distances from the shore (1, 25, and 50 m) at eight different freshwater lakes in Austria in June 2018. Water samples were filtered, and microorganisms were cultured on two different media types, TSA (a general growth medium) and KBC (a medium semi-selective for bacteria in the genus Pseudomonas). Mean concentrations (colony forming units per mL, or CFU/mL) of bacteria cultured on TSA ranged from 19,800 (Wörthersee) to 210,500 (Gosaulacke) CFU/mL, and mean concentrations of bacteria cultured on KBC ranged from 2590 (Ossiachersee) to 11,000 (Vorderer Gosausee) CFU/mL. There was no significant difference in sampling distance from the shore for concentrations of microbes cultured on TSA (p = 0.28). A wireless bathymetry sensor was tethered to the drone to map temperature and depth across the sampling domain of each of the lakes. At the 50 m distance from the shore, temperature ranged from 17 (Hinterer Gosausee, and Gosaulacke) to 26 °C (Wörthersee), and depth ranged from 2.8 (Gosaulacke) to 11.1 m (Grundlsee). Contour maps of concentrations of culturable bacteria across the drone sampling domain revealed areas of high concentrations (hot spots) in some of the lakes. The percentage of ice-nucleation active (ice+) bacteria cultured on KBC ranged from 0% (0/64) (Wörthersee) to 58% (42/72) (Vorderer Gosausee), with a mean of 28% (153/544) for the entire sample set. Future work aims to elucidate the structure and function of entire microbial assemblages within and among the Austrian lakes.

Diversity of Endophytic and Epiphytic Bacteria From Sugarcane in Khuzestan, Iran

Abstract Diverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Commercial formulations of bacteria antagonist to plant pathogenic microbes and ice nucleation active bacteria have been utilized as an environmentally safe method to manage plant disease and to prevent frost damage respectively. Bacteria were isolated from the leaf and sheath of sugarcane (CP69-1026 CP57-614, CP48-103, CP73-21, and CP70-1143 cultivars) verities grown in the field in Khuzestan province, Iran. Bacteria were found in both sheaths and leaves of sugarcane plants which they were significantly higher in density in leaves and which most were endophytic. The bacterial strains were 10 groups on the basis of the biochemical characteristic, which their 16S rRNA encoding gene from representatives were amplified and subjected to sequencing. Results of sequences analyze using blast software from the NCBI website and phylogenetic analysis showed that the representative strains belonged to a wide variety of phylogenetic groups. These results indicated that they were closely related to Burkholderia and Ralstonia from β-Proteobacteria, Mesorhizobium, Ochrobactrum, Sphingomonas from α-Proteobacteria, Microbacterium, Curtobacterium and Leifsonia from Actinobacteria and Xanthomonas from γ-Proteobacteria. This is the first report of the presence of endophytic and epiphytic bacteria from sugarcane in Khuzestan, Iran.

Freeze-Float Selection of Ice Nucleators.

In this manuscript, we developed a screening system that employs the difference in density between liquid water and ice (0.9998 g/cm3 vs 0.9168 g/cm3 at 0 °C) to identify ice-nucleating agents (INAs) that are encapsulated into droplets of water suspended in silicone oil of intermediate density (0.939 g/cm3). Droplets of liquid water stably reside at the interface of the silicone oil and perfluoro oil (1.6658 g/cm3); freezing causes the aqueous droplets to float to the top of the silicone oil layer. We demonstrated the feasibility of this screening system by using droplets that contained well-defined ice-nucleator Snomax. The droplets with and without Snomax froze at different temperatures and separated into two groups in our system. We employed the screening system to test samples that have different ice-nucleating activities. Starting from known ice-nucleating active bacteria Pseudomonas syringae, we confirmed that droplets that contain an increasing amount of ice-nucleating bacteria per droplet exhibit a dose-dependent increase in ice nucleation. When droplets containing different amounts of P. syringae were separated using a freeze-float setup, we observed that the droplets that floated at higher temperature contained more ice-nucleating active bacteria. The outlined system, thus, permits simple power-free separation of droplets that contain effective INA from those that contain weak or no INA. Such a setup can be used as a starting point for the development of high-throughput approaches for the discovery of new INAs.

Ice Nucleation Activity and Aeolian Dispersal Success in Airborne and Aquatic Microalgae.

Microalgae are common members of the atmospheric microbial assemblages. Diverse airborne microorganisms are known to produce ice nucleation active (INA) compounds, which catalyze cloud and rain formation, and thus alter cloud properties and their own deposition patterns. While the role of INA bacteria and fungi in atmospheric processes receives considerable attention, the numerical abundance and the capacity for ice nucleation in atmospheric microalgae are understudied. We isolated 81 strains of airborne microalgae from snow samples and determined their taxonomy by sequencing their ITS markers, 18S rRNA genes or 23S rRNA genes. We studied ice nucleation activity of airborne isolates, using droplet freezing assays, and their ability to withstand freezing. For comparison, we investigated 32 strains of microalgae from a culture collection, which were isolated from polar and temperate aqueous habitats. We show that ∼17% of airborne isolates, which belonged to taxa Trebouxiphyceae, Chlorophyceae and Stramenopiles, were INA. A large fraction of INA strains (over 40%) had ice nucleation activity at temperatures ≥-6°C. We found that 50% of aquatic microalgae were INA, but the majority were active at temperatures <-12°C. Most INA compounds produced by microalgae were proteinaceous and associated with the cells. While there were no deleterious effects of freezing on the viability of airborne microalgae, some of the aquatic strains were killed by freezing. In addition, the effect of desiccation was investigated for the aquatic strains and was found to constitute a limiting factor for their atmospheric dispersal. In conclusion, airborne microalgae possess adaptations to atmospheric dispersal, in contrast to microalgae isolated from aquatic habitats. We found that widespread taxa of both airborne and aquatic microalgae were INA at warm, sub-zero temperatures (>-15°C) and may thus participate in cloud and precipitation formation.

Ice Nucleation Active bacteria in Mount Lawu forest, Indonesia: 3. Isolation and estimation of bacterial populations on bryophyte

Abstract. Latifah NH, Susilowati A, Suratman. 2018. Ice Nucleation Active bacteria in Mount Lawu forest, Indonesia: 3. Isolation and estimation of bacterial populations on bryophyte. Asian J For 2: 67-75. This study aimed to isolate and estimate the Ice Nucleation Active (INA) bacteria population on bryophyte from the trekking route of Cemoro Sewu, Mount Lawu, Indonesia. Bryophyte samples were taken at three different altitudes, i.e., 2,000, 2,300, and 2,500 m above sea level (asl). Isolation of INA bacteria was carried out by the method of spread plate on King's B and nutrient agar plus 2.5% glycerol (NAG) medium. Bryophyte was identified by referring to the literature of Sutama (1995), Hasan and Ariyanti (2004), Ignatova and Samkova (2006), and Damayanti (2006). Ice nucleation activity was determined by the multiple-tube test method. The bacterial suspension tube was put into the circulating alcohol bath at a temperature of -10°C for 5 minutes. The number of INA bacteria on the bryophyte was estimated with the nucleation multiple tube method. The number (cell/g) of INA bacteria from the fresh weight of bryophyte was estimated based on the Most Probable Number (MPN) tables according to the formula of Thomas's 333 series. INA bacteria were identified through morphological and biochemical characters. Independent Sample T-Test analyzed the population of INA bacteria on terrestrial and epiphytes bryophytes with a significance of 5%. The results showed that 7 INA bacteria were isolated from the bryophyte Campylopus umbellatus (Arn.) Paris (Leucobryaceae, Musci). The population of INA bacteria in terrestrial bryophytes at each altitude were greater with 346 cell/g, 86 cell/g, and 396 cell/g, respectively, than that in epiphytes bryophyte with 50 cell/g, 50 cell/g, 176 cell/g, respectively.

Enumeration of ice nucleation active bacteria and severity of frost injury (embun upas) on potato in Wonosobo, Dieng Plateau

The altitude of the Dieng plateau is between 1,500-2,500 meters above sea level (m ASL), with an average temperature of 14°C. During the dry season, the temperature in this area becomes very cold that reaches -2°C, therefore frost injury may occur in potato, which is often referred to as embun upas (frozen dew). Frost injury causes the production of potatoes in Dieng to decline. Frost injury in plants is characterized by spots symptom, which can be brown, yellow, or black on leaves surface with the chlorotic ring that surrounds them. The phenomenon of frost injury is thought to be related to the activity of ice formation by the Ice Nucleation Active (INA) bacteria and triggered by cold temperatures (-2°C). This study aimed to determine the number of INA bacteria and its correlation with frost injury spots on potato leaves and to discover the class of INA bacteria based on ice formation temperature. The sampling of the potato leaves used purposive sampling method. Leaves showed frost injury symptom with different severity were taken from a different altitude of ± 2,000 m ASL (Dieng Wetan village) and ± 2,500 m ASL (Sembungan village). Bacterial isolation was done by spread plate method on Nutrient Agar supplemented with 2.5% glycerol. The activity of ice nucleation was determined using tube nucleation method. Estimation of INA bacteria number was done by multiple-tube nucleation method 3.3.3. Correlation between the number of INA bacteria and percentages of frost injury spots was analyzed using SPSS bivariate. The results showed that the highest INA bacteria number i.e. >2.75 × 104 was found on scale 3 of leave spots while the lowest value of 7.25 × 103 on scale 1. Correlation between the populations of INA bacteria with the scale of frost injury spots showed 0.844. The correlation values ranging from 0.80 to 1.00 indicating a strong association between the INA bacterial number and the scale of frost injury spots of potato leaves. Based on the ice forming temperatures, INA bacteria were classified into three classes, i.e., class A that can freeze water at temperatures of -2 to -4°C, class B -5 to -7°C and freezing point of class C below -8°C.

Ice nucleation active bacteria from pistachio in Kerman Province, Iran

Frost damage is one of the major factors threatening pistachio production in southern part of Iran. In the present study, an attempt was made to screen and characterize ice nucleation bacteria associated with frost damage among some predominant epiphytic microflora. A large number of epiphytic bacteria was isolated from different regions in Kerman province. 1200 bacterial isolates were obtained from bud, twig, leaf and green fruit surfaces. Sixty-four strains exhibited ice nucleation activity. Among these, a vast microbial diversity was observed. For preliminary characterization of the isolates, certain key biochemical and pathogenicity tests were performed. The preserved cultures were often checked for ice formation and loss of ice nucleation activity was found in most cultures. Furthermore, factors affecting the increment of ice nucleation activity were studied and the isolates that recovered their ice nucleation activity were selected for sequence analyses of 16SrDNA, rpoD and recA genes, then compared with other isolates in NCBI GenBank using blasts methods. Pseudomonas fragi, P. putida, P. moraviensis, P. virdiflava and Pantoea agglomerans were the major predominant ice nucleation active bacteria established on plant surfaces. This work reports for the first time the presence of two ice nucleation active bacteria, P. fragi and P. moraviensis, on pistachio plants.