Evaluation of lettuce germplasm resistance to gray mold disease for organic cultivations.

This study was conducted to evaluate the resistance of 212 accessions of lettuce germplasm to gray mold disease caused by Botrytis cinerea. The lettuce germplasm were composed of five species: Lactuca sativa (193 accessions), L. sativa var. longifolia (2 accessions), L. sativa var. crispa (2 accessions), L. saligna (2 accessions), and L. serriola (1 accession); majority of these originated from Korea, Netherlands, USA, Russia, and Bulgaria. After 35 days of spray inoculation with conidial suspension (3×10(7) conidia/ml) of B. cinerea on the surface of lettuce leaves, tested lettuce germplasm showed severe symptoms of gray mold disease. There were 208 susceptible accessions to B. cinerea counted with 100% of disease incidence and four resistant accessions, IT908801, K000598, K000599, and K021055. Two moderately resistant accessions of L. sativa, K021055 and IT908801, showed 20% of disease incidence of gray mold disease at 45 days after inoculation; and two accessions of L. saligna, K000598 and K000599, which are wild relatives of lettuce germplasm with loose-leaf type, showed complete resistance to B. cinerea. These four accessions are candidates for breeding lettuce cultivars resistant to gray mold disease.

Evaluation of Watermelon Germplasm for Resistance to Phytophthora Blight Caused by Phytophthora capsici.

This study was conducted to determine the Phytophthora rot resistance of 514 accessions of watermelon germplasm, Citrullus lanatus var lanatus. About 46% of the 514 accessions tested were collections from Uzbekistan, Turkey, China, U.S.A., and Ukraine. Phytophthora capsici was inoculated to 45-day-old watermelon seedlings by drenching with 5 ml of sporangial suspension (10(6) sporangia/ml). At 7 days after inoculation, 21 accessions showed no disease symptoms while 291 accessions of susceptible watermelon germplasm showed more than 60.1% disease severity. A total of 510 accessions of watermelon germplasm showed significant disease symptoms and were rated as susceptible to highly susceptible 35 days after inoculation. The highly susceptible watermelon germplasm exhibited white fungal hyphae on the lesion or damping off with water-soaked and browning symptoms. One accession (IT032840) showed moderate resistance and two accessions (IT185446 and IT187904) were resistant to P. capsici. Results suggest that these two resistant germplasm can be used as a rootstock and as a source of resistance in breeding resistant watermelon varieties against Phytophthora.

Larkinella bovis sp. nov., isolated from fermented bovine products, and emended descriptions of the genus Larkinella and of Larkinella insperata Vancanneyt et al. 2006.

A novel bacterial strain, designated M2T2B15(T), was isolated from fermented bovine products and was characterized by using a polyphasic approach. Colonies were reddish pink and circular with entire margins. Cells were strictly aerobic, Gram-reaction-negative, oxidase- and catalase-positive rods that lacked flagella and were motile by gliding. Flexirubin-type pigments were absent. 16S rRNA gene sequence analysis indicated that strain M2T2B15(T) was related most closely to Larkinella insperata LMG 22510(T) (94.4 % similarity) but shared <87 % similarity with other members of the phylum Bacteroidetes. The major cellular fatty acids were C(16 : 1)ω5c, iso-C(15 : 0) and iso-C(17 : 0) 3-OH. The polar lipids were phosphatidylethanolamine, phosphatidylserine, two unidentified aminophospholipids and two unidentified polar lipids. Menaquinone 7 (MK-7) was the major respiratory quinone. The G+C content of the DNA of strain M2T2B15(T) was 52 mol%. The phenotypic, genotypic and phylogenetic data presented clearly indicate that strain M2T2B15(T) represents a novel species of the genus Larkinella, for which the name Larkinella bovis sp. nov. is proposed. The type strain is M2T2B15(T) (=KACC 14040(T) =NBRC 106324(T)). Emended descriptions of the genus Larkinella and of Larkinella insperata Vancanneyt et al. 2006 are also proposed.

Dyella thiooxydans sp. nov., a facultatively chemolithotrophic, thiosulfate-oxidizing bacterium isolated from rhizosphere soil of sunflower (Helianthus annuus L.).

A Gram-negative, aerobic, motile, rod-shaped, thiosulfate-oxidizing bacterium, designated ATSB10(T), was isolated from rhizosphere soil of sunflower (Helianthus annuus L.). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain ATSB10(T) was closely related to members of the genera Dyella (96.4-98.1 % 16S rRNA gene sequence similarity) and Luteibacter (96.4-97.0 %) and Fulvimonas soli LMG 19981(T) (96.7 %) and Frateuria aurantia IFO 3245(T) (97.8 %). The predominant fatty acids were iso-C(16 : 0), iso-C(17 : 1)ω9c and iso-C(15 : 0). The major quinone was Q-8. The G+C content of the genomic DNA was 66.0 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidyldimethylethanolamine, an unknown phospholipid, unknown aminophospholipids and an unknown aminolipid. On the basis of phenotypic properties, phylogenetic distinctiveness and DNA-DNA relatedness, strain ATSB10(T) represents a novel species in the genus Dyella, for which the name Dyella thiooxydans sp. nov. is proposed. The type strain is ATSB10(T) (=KACC 12756(T) =LMG 24673(T)).

Pandoraea thiooxydans sp. nov., a facultatively chemolithotrophic, thiosulfate-oxidizing bacterium isolated from rhizosphere soils of sesame (Sesamum indicum L.).

A facultatively chemolithoautotrophic, thiosulfate-oxidizing, Gram-negative, aerobic, motile, rod-shaped bacterial strain, designated ATSB16(T), was isolated from rhizosphere soils of sesame (Sesamum indicum L.). 16S rRNA gene sequence analysis demonstrated that this strain was closely related to Pandoraea pnomenusa LMG 18087(T) (96.7 % similarity), P. pulmonicola LMG 18016(T) (96.5 %), P. apista LMG 16407(T) (96.2 %), P. norimbergensis LMG 18379(T) (96.1 %) and P. sputorum LMG 18819(T) (96.0 %). Strain ATSB16(T) shared 96.0-96.4 % sequence similarity with four unnamed genomospecies of Pandoraea. The major cellular fatty acids of the strain ATSB16(T) were C(17 : 0) cyclo (33.0 %) and C(16 : 0) (30.6 %). Q-8 was the predominant respiratory quinone. The major polar lipids were phosphatidylmethylethanolamine, diphosphatidylglycerol, phosphatidylethanolamine and two unidentified aminophospholipids. Hydroxyputrescine and putrescine were the predominant polyamines. The genomic DNA G+C content of the strain was 64.0 mol%. On the basis of the results obtained from this study, strain ATSB16(T) represents a novel species of the genus Pandoraea, for which the name Pandoraea thiooxydans sp. nov. is proposed. The type strain is ATSB16(T) (=KACC 12757(T) =LMG 24779(T)).

Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov.

A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13(T) was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13(T) was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13(T) exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13(T) was most closely related to Burkholderia kururiensis KP23(T) (98.7%), Burkholderia tuberum STM678(T) (96.5%) and Burkholderia phymatum STM815(T) (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C(18:1) omega7c (28.22%), C(16:1) omega7c/15 iso 2OH (15.15%), and C(16:0) (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13(T) within the genus Burkholderia. Though the strain ATSB13(T) shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA-DNA hybridization between strain ATSB13(T) and B. kururiensis KP23(T) was 100%, and hence, it is inferred that strain ATSB13(T) is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23(T) = JCM 10599(T) = DSM 13646(T)) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13(T) = KACC 12758(T)).

Thiosulfate Oxidation and mixotrophic growth of Methylobacterium goesingense and Methylobacterium fujisawaense.

The mixotrophic growth with methanol plus thiosulfate was examined in nutrient-limited mixotrophic condition for Methylobacterium goesingense CBMB5 and Methylobacterium fujisawaense CBMB37. Thiosulfate oxidation increased the growth and protein yield in mixotrophic medium that contained 150 mM methanol and 20 mM sodium thiosulfate, at 144 h. Respirometric study revealed that thiosulfate was the most preferable reduced inorganic sulfur source, followed by sulfite and sulfur. M. goesingense CBMB5 and M. fujisawaense CBMB37 oxidized thiosulfate directly to sulfate, and intermediate products of thiosulfate oxidation such as polythionates, sulfite, and sulfur were not detected in spent medium and they did not yield positive amplification for tested soxB primers. Enzymes of thiosulfate oxidation such as rhodanese and sulfite oxidase activities were detected in cell-free extracts of M. goesingense CBMB5, and M. fujisawaense CBMB37, and thiosulfate oxidase (tetrathionate synthase) activity was not observed. It indicated that both the organisms use the "non-S4 intermediate" sulfur oxidation pathway for thiosulfate oxidation. It is concluded from this study that M. goesingense CBMB5, and M. fujisawaense CBMB37 exhibited mixotrophic metabolism in medium containing methanol plus thiosulfate and that thiosulfate oxidation and the presence of a "Paracoccus sulfur oxidation" (PSO) pathway in methylotrophic bacteria are species dependant.

Chemolithoautotrophic oxidation of thiosulfate and phylogenetic distribution of sulfur oxidation gene (soxB) in rhizobacteria isolated from crop plants.

Twenty-one thiosulfate-oxidizing bacteria were isolated from rhizosphere soils and 16S rRNA analysis revealed that the isolates were affiliated with seven different phylogenetic groups within the Beta and Gamma subclasses of Proteobacteria and Actinobacteria. Among these, five genera, including Dyella, Burkholderia, Alcaligenes, Microbacterium and Leifsonia sp., represented new sulfur oxidizers in rhizosphere soils. The thiosulfate-oxidizing Dyella, Burkholderia, Alcaligenes, Microbacterium, Leifsonia and Pandoraea were able to grow chemolithotrophically with a medium containing thiosulfate and exhibited growth coupled with thiosulfate oxidation. They accumulated intermediate products such as sulfur, sulfite and trithionate in the spent medium during the time course of thiosulfate oxidation, and these products were finally oxidized into sulfate. Furthermore, they possessed thiosulfate-metabolizing enzymes such as rhodanese, thiosulfate oxidase, sulfite oxidase and trithionate hydrolase, suggesting that these bacteria use the 'S4 intermediate' (S4I) pathway for thiosulfate oxidation. Phylogenetic analysis of the soxB gene revealed that Pandoraea sp. and Pandoraea pnomenusa strains formed a separate lineage within Betaproteobacteria.

Soft Rot of Eggplant (Solanum melongena) Caused by Choanephora cucurbitarum in Korea.

In April 2002 and 2003, soft rot on fruit of eggplant (Solanum melongena) caused by Choanephora cucurbitarum was observed in the experimental fields at Gyeongnam Agricultural Research and Extension Services in Korea. The disease began with water-soaking and dark-green lesions, and then the infected tissues were rapidly rotten. Sporangium was subglobose in shape and sized 40~130 µm. Monosporous sporangiola were elliptic, fusiform or ovoid, brown in color, and measured as 12~20 × 6~14 µm. Sporangiospores having three or more appendages were elliptic, fusiform or ovoid in shape, dark brown or brown in color, and sized 14~20 × 7~16 µm. The fungus grew well on potato dextrose agar between 15 and 40℃ and its optimum growth temperature was 30℃. Based on morphological characteristics, the causal fungus of the fruit soft rot of eggplant was identified as C. cucurbitarum. This is the first report on the soft rot of S. melongena caused by C. cucurbitarum in Korea.

Phytophthora cactorum can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.

Phytophthora cactorum grown on basal agarose medium or in liquid basal medium produced oospores after being transferred to water agarose. The numbers of oospores produced under such conditions depended on the age of the culture prior to exposure to nutrient deprivation. When the concentration of basal medium used for cultivation of P. cactorum was increased, the numbers of oospores produced after being transferred to water agarose was also increased. P. cactorum grown on basal agarose medium also produced oospores when its mycelial growth was restricted after reaching the edge of Petri plates. In 5 cm plates oospore formation occurred in the third week, whereas in 9 cm and 14 cm plates oospores appeared in the fourth week. Most oospores were formed near the edge of the plates. The non-saponifiables extracted from mycelia of P. cactorum grown in liquid basal medium were stimulatory to oospore formation by P. cactorum and Phytophthora parasitica, whereas the saponifiables were stimulatory to P. cactorum only. Extracts from culture filtrate and basal medium were not stimulatory to oospore formation by either fungus. When the non-saponifiables were fractionated by Florisil columm chromatography, only fraction 1 was not active. Fractions 2, 3 and 4 were stimulatory to oospore formation by both P. cactorum and P. parasitica. These results support the hypothesis that P. cactorum, and possibly other pythiaceous fungi as well, can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.

Stimulation of sexual reproduction in Phytophthora cactorum by phospholipids is not due to sterol contamination.

Phytophthora cactorum did not form oospores on basal medium unless phosphatidylcholine (lecithin) or phosphatidylethanolamine (cephalin) was added. After removal of putative sterols by aminopropyl column chromatography, the activities of lecithin and cephalin were increased 47- and 2.8-fold, respectively, thus confirming the previous reports that sterols are not essential for sexual reproduction in this organism. Thin-layer chromatography (TLC) of the commercial lecithin revealed the presence of an unknown inhibitory substance which, when added to the purified lecithin, caused a 50% reduction of oospore formation. Commercial cephalin also showed a twofold increase in activity after removal of putative sterols and the existence of an unknown inhibitor when it was subjected to TLC. Addition of the inhibitor to the purified cephalin completely inhibited the growth of the test organism. One sample of lecithin tested was not stimulatory to oospore formation. However, after washing with deionized water or NaCl solution, it induced the production of 17300 and 24450 oospores (100 µg)-1, respectively. The ability of cephalin to induce oospore formation was increased 2⋅3-fold by washing with deionized water and 8⋅3-fold by washing with NaCl solution. Like sterols, the digitonin precipitable component (digitonide) of the non-phospholipid fraction of commercial lecithin or cephalin was stimulatory to oospore formation of P. cactorum but not Phytophthora parasitica. However, the non-digitonide component was not only more active than the digitonide component, but also stimulatory to P. parasitica. Gas chromatography and mass spectrometry (GC-MS) analysis of the digitonide component from lecithin failed to detect any putative sterol contaminant. The amount of the putative sterol contaminant in the digitonide component from cephalin was also below the detection limit of GC-MS. When 0.01-10 ng cholesterol was added to basal medium discs each containing 100 fig cephalin, the numbers of oospores produced by P. cactorum and P. parasitica were not significantly changed. It is concluded that, in the fungi tested, sterols did not play any significant role in the stimulation of sexual reproduction by highly purified phospholipids.