Identification and Characterization of Pseudomonas syringae pv. syringae,a Causative Bacterium of Apple Canker in Korea

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of −0.7°C, indicating an INA⁺ bacterium. The ice-nucleating temperature was −4.7°C for a non-treated control (sterilized distilled water), whereas it was −9.6°C for an INA⁻ bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.     

Distribution, Population Dynamics, and Characteristics of Ice Nucleation-Active Bacteria in Deciduous Fruit Tree Orchards

Deciduous fruit tree orchards located in the Pacific Northwest were surveyed over a 3-year period for the presence of ice nucleation-active (INA) bacteria. In the Yakima Valley, only about 30% of the fruit tree orchards contained INA bacteria (median population ca. 3 × 10² CFU/g [fresh weight]) in contrast to nearly 75% of the orchards in the Hood River Valley (median population ca. 5 × 10³ CFU/g [fresh weight]). These INA populations ranged from less than 10 to over 10⁶ CFU/g (fresh weight) of blossoms and, in Hood River Valley orchards, generally comprised over 10% of the total bacterial population. Populations of INA bacteria fluctuated during the year with highest levels developing on buds and flowers during the cool, wet spring, followed by a drop in populations during the warmer, drier, summer months and finally a gradual increase in the autumn. The INA bacteria persisted on dormant buds from which they again colonized young developing vegetative tissues. All INA bacteria were identified as Pseudomonas syringae. The frequency of ice nucleation at −5°C for these strains ranged from nearly every cell being INA to less than 1 in 10⁷ cells. The median frequency of ice nucleation at −5°C was 10⁴ cells per ice nucleus. The INA P. syringae strains from individual orchards were diverse with respect to bacteriocin typing and in ice nucleation frequency. The consistent absence of detectable INA bacteria or presence of low populations in most of the orchards surveyed during periods when critical temperatures (i.e., −2 to −5°C) were common indicated a limited role for INA bacteria in frost susceptibility of most Pacific Northwest orchards.     

Assessment of Parasitic Activity of Fusarium Strains Obtained from a Heterodera schachtii-Suppressive Soil

This study assessed the potential impact of various Fusarium strains on the population development of sugarbeet cyst nematodes. Fungi were isolated from cysts or eggs of Heterodera schachtii Schmidt that were obtained from a field suppressive to that nematode. Twenty-six strains of Fusarium spp. were subjected to a phylogenic analysis of their rRNA-ITS nucleotide sequences. Seven genetically distinct Fusarium strains were evaluated for their ability to influence population development of H. schachtii and crop performance in greenhouse trials. Swiss chard (Beta vulgaris) seedlings were transplanted into fumigated field soil amended with a single fungal strain at 1,000 propagules/g soil. One week later, the soil was infested with 250 H. schachtii J2/100 cm³ soil. Parasitized eggs were present in all seven Fusarium treatments at 1,180 degree-days after fungal infestation. The percentage of parasitism ranged from 17 to 34%. Although the most efficacious F. oxysporum strain 471 produced as many parasitized eggs as occurred in the original suppressive soil, none of the Fusarium strains reduced the population density of H. schachtii compared to the conducive check. This supports prior results that Fusarium spp. were not the primary cause of the population suppression of sugarbeet cyst nematodes at this location.     

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.     

Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit,Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis

The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level.     

Identification and Characterization of Fungal Pathogens Associated with Boxwood Diseases in the Republic of Korea

Boxwood is a representative ornamental shrub that is widely used in landscaping horticulture. After pruning, damaged leaves or stems of boxwoods are unavoidably vulnerable to infection by various plant pathogens. Several boxwood diseases caused by fungi, such as Volutella blight and Macrophoma leaf spot, have been reported worldwide including Republic of Korea. In this study, we isolated and identified fungal pathogens of boxwood diseases that occurred in Korea and characterized their morphological and taxonomic characteristics. Boxwood samples showing blight symptoms were collected in Seoul, Republic of Korea, and the putative fungal pathogens Pseudonectria buxi, P. foliicola, and Neofusicoccum buxi were successfully identified. Investigation of the morphological features of the field isolates, including mycelial growth and conidial morphology, and phylogenetic analysis of multiple DNA barcode loci revealed that there were some morphological and genetic variations among isolates, but all of the analyzed isolates were closely related to the corresponding reference strains. We also found that P. foliicola strains were more virulent than P. buxi, and the N. buxi strains isolated in this study were weak pathogens or saprophytes. The results of our study will contribute to the development of control strategies for boxwood diseases caused by fungi and accelerate research on the complex ecology of boxwood diseases.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

Efficacy of Pseudomonas syringae in the management of potato tuber diseases in storage

Silver scurf caused by Helminthosporium solani and dry rot caused by Fusarium spp. are tuber diseases of economic importance in potato-growing areas worldwide. Recently, the two pathogens have developed resistance to thiabendazole (TBZ), a post-harvest fungicide commonly used for their control. Therefore, alternative disease control strategies are needed. The present study assessed the efficacy of the biopesticides Bio-Save 10LP (Pseudomonas syringae-strain ESC-10; Ps10) and Bio-Save 11LP (P. syringae-strain ESC-11; Ps11) against silver scurf and dry rot. Approximately 30 isolates representing the genus Fusarium were obtained from symptomatic potato specimens with dry rot from New Brunswick (NB), Nova Scotia (NS), Prince Edward Island (PE) and Alberta (AB), Canada. Species isolated were Fusarium sambucinum, Fusarium tumidum, Fusarium coeruleum, Fusarium culmorum, and Fusarium avenaceum. H. solani isolated from AB, NB and PE was included in the study as the causal agent of silver scurf. The efficacy of P. syringae against F. sambucinum and H. solani was tested in vitro. Ps10 and Ps11 inhibited the growth of H. solani up to 68% (NB isolate) and 73% (PE isolate), respectively and the inhibition was more or less comparable with that of TBZ. F. sambucinum was not significantly inhibited by Ps10; however Ps11 significantly inhibited AB, PE and NB isolates by 43%, 28% and 54%, respectively. Conversely, TBZ inhibited AB, PE and NB isolates of Fusarium spp. in vitro by 86%, 88% and 100%, respectively. TBZ in combination with either Ps10 or Ps11 did not always reduce the growth of H. solani or Fusarium spp. in vitro compared to that of TBZ alone. Storage trials conducted in NB and PE assessed the efficacy of P. syringae against H. solani or Fusarium spp. in vivo and confirmed that the application of P. syringae or TBZ alone or in combination significantly reduced the incidence and/or severity of silver scurf and Fusarium dry rot. Ps11 alone or in combination with TBZ was significantly more effective than Ps10 in controlling silver scurf disease severity. The reduction in disease severity of dry rot and silver scurf in storage due to Ps10, Ps11, or TBZ or their combinations was consistently comparable. Results indicate that the use of P. syringae (strains ESC-11 or ESC-10) as a post-harvest treatment can contribute to the management of both silver scurf and Fusarium dry rot in potato storages.     

Fumonisin and T-2 toxin production of Fusarium spp. isolated from complete feed and individual agricultural commodities used in shrimp farming

Fusarium spp. are plant pathogens producing fumonisins and trichothecenes that both affect human and animal health. In the present study, 40 fungal strains were isolated and species identified from 35 shrimp feed samples and from 61 agricultural raw materials. F. verticillioides was the predominant species (85 %) mostly found in corn and soybean meal, while no Fusarium contamination was detected in shrimp feed. Levels of 10 % of F. oxysporum were isolated from peanut and 5 % of F. equiseti contamination in corn and peanut. To determine the ability of toxin production, enzyme-linked immunosorbent assay, polymerase chain reaction, and ultra-pressure liquid chromatography-tandem mass spectrometry were performed. All but four of the fumonisin-producing strains contained the FUM1 gene. No Fusarium synthesized T-2 toxin nor contained the Tri5 gene. This survey brings more data on mycotoxin contamination in the food chain of animal feed production, and leads to the awareness of the use of contaminated raw materials in shrimp farming.     

Genetic Characterization of Pseudomonas syringae pv. syringae Strains from Stone Fruits in California

Strains of Pseudomonas syringae pv. syringae were isolated from healthy and diseased stone fruit tissues sampled from 43 orchard sites in California in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and the presence of the syrB and syrC genes and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. The 19 strains of P. syringae pv. syringae examined by restriction fragment length polymorphism analysis contained the syrB and syrC genes, whereas no hybridization occurred with 4 strains of other P. syringae pathovars. The P. syringae pv. syringae strains from stone fruit, except for a strain from New Zealand, generated ERIC genomic fingerprints which shared four fragments of similar mobility. Of the P. syringae pv. syringae strains tested from other hosts, only strains from rose, kiwi, and pear generated genomic fingerprints that had the same four fragments as the stone fruit strains. Analysis of the ERIC fingerprints from P. syringae pv. syringae strains showed that the strains isolated from stone fruits formed a distinct cluster separate from most of the strains isolated from other hosts. These results provide evidence of host specialization within the diverse pathovar P. syringae pv. syringae.     

Evolution of the Core Genome of Pseudomonas syringae, a Highly Clonal, Endemic Plant Pathogen

Pseudomonas syringae is a common foliar bacterium responsible for many important plant diseases. We studied the population structure and dynamics of the core genome of P. syringae via multilocus sequencing typing (MLST) of 60 strains, representing 21 pathovars and 2 nonpathogens, isolated from a variety of plant hosts. Seven housekeeping genes, dispersed around the P. syringae genome, were sequenced to obtain 400 to 500 nucleotides per gene. Forty unique sequence types were identified, with most strains falling into one of four major clades. Phylogenetic and maximum-likelihood analyses revealed a remarkable degree of congruence among the seven genes, indicating a common evolutionary history for the seven loci. MLST and population genetic analyses also found a very low level of recombination. Overall, mutation was found to be approximately four times more likely than recombination to change any single nucleotide. A skyline plot was used to study the demographic history of P. syringae. The species was found to have maintained a constant population size over time. Strains were also found to remain genetically homogeneous over many years, and when isolated from sites as widespread as the United States and Japan. An analysis of molecular variance found that host association explains only a small proportion of the total genetic variation in the sample. These analyses reveal that with respect to the core genome, P. syringae is a highly clonal and stable species that is endemic within plant populations, yet the genetic variation seen in these genes only weakly predicts host association.     

Biodegradation of a mixture of PAHs by non-ligninolytic fungal strains isolated from crude oil-contaminated soil

Nine native non-ligninolytic fungal strains were isolated from Maya crude oil-contaminated soil and selected based on their ability to grow and use crude oil and several polycyclic aromatic hydrocarbons (PAHs) as carbon source, for their application to PAH removal in soil. The fungi were identified by PCR amplification of intergenic transcribed sequences regions and microbiological techniques, and results showed them to be part of the genera Fusarium, Neurospora, Aspergillus, Scedosporium, Penicillium, Neosartorya and Talaromyces. A primary selection of fungi was made in minimal medium plates, considering the tolerance to different concentrations of PAHs for each strain. The radial extension rate exhibited significant differences (p < 0.05) from 200 to 1,000 mg of PAHs mixture l^?1. A secondary selection of Aspergillus terreus, Talaromyces spectabilis, and Fusarium sp. was achieved based on their tolerance to 2,000 mg of a mixture of Phenanathrene and Pyrene kg^?1 of soil in a solid-state microcosm system for 2 weeks. The percentage of PAH removal obtained by the three strains was approximately 21 % of the mixture.     

Expression and localization of an ice nucleating protein from a soil bacterium, Pseudomonas borealis

An ice nucleating protein (INP) coding region with 66% sequence identity to the INP of Pseudomonas syringae was previously cloned from P. borealis, a plant beneficial soil bacterium. Ice nucleating activity (INA) in the P. borealis DL7 strain was highest after transfer of cultures to temperatures just above freezing. The corresponding INP coding sequence (inaPb or ina) was used to construct recombinant plasmids, with recombinant expression visualized using a green fluorescent protein marker (gfp encoding GFP). Although the P. borealis strain was originally isolated by ice-affinity, bacterial cultures with membrane-associated INP-GFP did not adsorb to pre-formed ice. Employment of a shuttle vector allowed expression of ina-gfp in both Escherichia coli and Pseudomonas cells. At 27 °C, diffuse fluorescence appeared throughout the cells and was associated with low INA. However, after transfer of cultures to 4 °C, the protein localized to the poles coincident with high INA. Transformants with truncated INP sequences ligated to either gfp, or an antifreeze protein-gfp fusion showed that the repetitive ice-nucleation domain was not necessary for localization. Such localization is consistent with the flanking residues of the INP associating with a temperature-dependent secretion apparatus. A polar location would facilitate INP–INP interactions resulting in the formation of larger aggregates, serving to increase INA. Expression of INPs by P. borealis could function as an efficient atmospheric dispersal mechanism for these soil bacteria, which are less likely to use these proteins for nutrient procurement, as has been suggested for P. syringae.     

Biodegradation of phenol by a filamentous fungi isolated from industrial effluents—identification and degradation potential

Thirty filamentous fungal strains were isolated from effluents of a stainless steel industry (Minas Gerais, Brazil) and tested for phenol tolerance. Fifteen strains of the genera Fusarium sp., Aspergillus sp., Penicillium sp. and Graphium sp. tolerants up to 10 mM of phenol were selected and tested for their ability to degrade phenol. Phenol degradation was a function of strain, time of incubation and initial phenol concentration. FIB4, LEA5 and AE2 strains of Graphium sp. and FE11 of Fusarium sp. presented the highest percentage phenol degradation, with 75% degradation of 10 mM phenol in 168 h for FIB4. A higher starting cell density of Graphium sp. FIB4 lead to a decrease in the time needed for full phenol degradation and increased the phenol degradation rate. All strains exhibited activity of catechol 1,2-dioxygenase and phenol hydroxylase in free cell extracts obtained from cells grown on phenol, suggesting that catechol was oxidized by the ortho type of ring fission. These data reported demonstrate the prospect after the application of filamentous fungal strains in protecting the environment from phenol pollution.     

New antibiotics produced by Bacillus subtilis strains

Two Bacillus subtilis strains isolated from the fruiting body of a basidiomycete fungus Pholiota squarrosa exhibited a broad range of antibacterial activity, including those against methicillin-resistant Staphylococcus aureus INA 00761 (MRSA) and Leuconostoc mesenteroides VKPM B-4177 resistant to glycopeptide antibiotics, as well as antifungal activity. The strains were identified as belonging to the “B. subtilis” complex based on their morphological and physiological characteristics, as well as by sequencing the 16S rRNA gene fragments. Both strains (INA 01085 and INA 01086) produced insignificant amounts of polyene antibiotics (hexaene and pentaene, respectively). Strain INA 01086 also produced a cyclic polypeptide antibiotic containing Asp, Gly, Leu, Pro, Tyr, Thr, Trp, and Phe, while the antibiotic of strain INA 01085 contained, apart from these, two unidentified nonproteinaceous amino acids. Both polypeptide antibiotics were new compounds efficient against gram-positive bacteria and able to override the natural bacterial antibiotic resistance.     

A Brevibacillus sp. antagonistic to mycotoxigenic Fusarium spp.

Antagonistic microbes were isolated from soils to control mycotoxin contamination of cereals by limiting the growth of mycotoxigenic Fusarium species. In total, 341 bacterial isolates were examined for antifungal activity against eight mycotoxigenic Fusarium species using dual culture assays. The screening identified 11 isolates that inhibited mycelial growth of all Fusarium species tested. The culture filtrates of 2 of the 11 isolates completely inhibited germination of conidia up to 21 days of incubation. These two isolates exhibited identical activity toward the fungi tested and were identified as Brevibacillus spp. based on 16S rRNA sequence homology. The most closely related species based on phylogenetic analysis was Brevibacillus reuszeri. Additional dual culturing using further fungal species showed that the antagonistic Brevibacillus inhibited the growth of most Fusarium species tested (39 of 46 species), two Epicoccum spp., one Alternaria sp., three Aspergillus spp. (3 of 11), and three Penicillium spp. (3 of 8). The in vivo assay was performed to test the efficacy of antagonistic Brevibacillus isolates on maize ears and revealed that the application of microbes suppressed ear rot (ANOVA, p = 0.0020). This Brevibacillus sp. may be an antagonist of the majority of Fusarium species, including mycotoxigenic species.     

Environmental Isolates of Fungi from Aquarium Pools Housing Killer Whales (Orcinus orca)

Systemic mycoses in killer whales (Orcinus orca) are rare diseases, but have been reported. Two killer whales died by fungal infections at the Port of Nagoya Public Aquarium in Japan. In this study, the fungal flora of the pool environment at the aquarium was characterized. Alternaria spp., Aspergillus spp. (A. fumigatus, A. niger, A. versicolor), Fusarium spp. and Penicillium spp. were isolated from the air and the pool surroundings. The other isolates were identified as fungal species non-pathogenic for mammals. However, the species of fungi isolated from the environmental samples in this study were not the same as those isolated from the cases of disease in killer whales previously reported.     

Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.     

Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury

Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately −5°C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log₁₀ mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of −2.5°C and −3.0°C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at −5°C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants.