Injection–Infiltration of Attached Grapefruit with Xanthomonas citri subsp. citri to Evaluate Seasonal Population Dynamics in Citrus Canker Lesions

To study population dynamics of Xanthomonas citri subsp. citri (Xcc) in citrus canker lesions on fruit, a needle‐free injector was used for infiltration of bacterial inoculum into fruit in situ on mature ‘Ruby Red’ grapefruit (Citrus paradisi Macf.) trees in Florida. Inoculations of Xcc at 10⁵ colony‐forming units (cfu) per ml were conducted in 2012 and 2013 on attached fruit varying from 15 mm to 90 mm in diameter. Inoculations were repeated every 2–3 weeks until the fruit were no longer injectable. On fruit less than 40 mm in size, erumpent lesions formed within 2 weeks of inoculation and expanded 1–9 mm in diameter from 30 to 120 days postinoculation (dpi). Xcc populations in lesions were 6–8 log cfu per lesion at 30 dpi and maintained this population up to 90 dpi. By 120 dpi, Xcc populations declined 1–3 log units as rainfall and temperature decreased in September–October. Xcc populations declined to ~3 log cfu per lesion after 120 dpi in November 2012 and 2013, whereas the population resurged to 5 log cfu per lesion after 180 dpi in January–February 2014.     

Production of yoghurt with mild taste by a Lactobacillus delbrueckii subsp. bulgaricus mutant with altered proteolytic properties

In this communication, we describe the isolation of a Lactobacillus delbrueckii subsp. bulgaricus 92063 mutant strain named pH-P11, which differed from the parent strain by low proteolytic activity and altered regulation of expression of lacZ in the presence of glucose or lactose. In the presence of lactose, beta-galactosidase activity was approximately twice as high in pH-P11 than in the wild type. pH-P11 exhibited protosymbiosis together with Streptococcus thermophilus. Yoghurt produced with pH-P11 was characterized by low acidity and little post-acidification during storage. The organoleptic properties (absence of bitterness and other off-flavors, weak sourness, and clear yoghurt taste) were those of a typical "yoghurt mild". This mild flavor was achieved at rather high cell counts of lactobacilli even at the end of shelf-life. High cell counts in conjunction with high beta-galactosidase activity make pH-P11 an interesting strain for application in yoghurt especially designed for consumers with lactose malabsorption. In contrast to "yoghurt mild", which is predominantly produced with Lactobacillus acidophilus together with Streptococcus thermophilus, the product obtained by fermentation with pH-P11 and Streptococcus thermophilus concurs with international standards for yoghurt. During frequent sub-culturing, strain pH-P11, which is supposed to differ from the wild type by one or a few so-far-not-characterized mutations, showed sufficient stability for application in industrial production.     

The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L⁻¹ of BA and 0.036 mg L⁻¹ of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L⁻¹ IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.     

野生秀丽四照花种子生物学特性及萌发研究

对野生秀丽四照花(Cornus hongkongensis subsp. elegans)种子的生物学特性进行观察测定,比较变温、低温层积、化学处理对种子萌发的影响。结果表明,秀丽四照花种子千粒重76.91 g,长5.81 mm、宽5.10 mm、厚3.99 mm。种子在整个试验过程中吸水率低于8%,说明有吸水性障碍的坚硬种皮抑制了种子萌发。变温条件下,温差较大的30 ℃/15 ℃处理对秀丽四照花种子萌发有较好的促进作用,萌发时滞短,萌发率和发芽势较高。秀丽四照花种子经30% NaOH浸泡10 min,可明显缩短萌发时间,但4 ℃低温层积处理对种子萌发没有显著效果。     

Characterization of the replicon of a 51-kb native plasmid from the gram-positive bacterium Leifsonia xyli subsp. cynodontis

The 4992-bp replicon of a large cryptic plasmid in the gram-positive bacterium Leifsonia xyli subsp. cynodontis was identified and sequenced. The replicon encoded two proteins essential for plasmid replication and stability. The putative replication protein (RepA) is homologous to that of the plasmids in mycobacterial pLR7 family, while the putative ParA protein immediately downstream of RepA is significantly homologous to the Walker-type ATPase required for partition of plasmid and chromosome of the gram-positive bacteria. These two proteins and other ORFs are clustered with the putative promoters and other regulatory sequences, illustrating an efficient organization of the replicon for this novel plasmid.     

Comparison of contamination and growth of Mycobacterium avium subsp. paratuberculosis on two different media

AIMS: The purpose of the study was to compare the growth of Mycobacterium avium subsp. paratuberculosis (Map) and the degree of contamination on Herrold's egg yolk medium (HEYM) and modified L?wenstein-Jensen medium (LJ). METHODS AND RESULTS: Culture of 2513 faecal samples from dairy cows was performed on each of the two media. The media were read after 5, 8 and 12 weeks of incubation. Overall, the proportion of contaminated samples was significantly higher on LJ (14.2%) than on HEYM (13.2%) after 12 weeks but the degree of contamination was slightly less on LJ. After 8 weeks of incubation, only 1.0% of the samples were Map positive in LJ with 4.9% on HEYM. After 12 weeks of incubation, 3.3% of the samples were Map positive in LJ whereas 6.9% were positive in HEYM. All suspect and culture positive samples were confirmed by IS900 PCR. CONCLUSIONS: HEYM supported growth of Map significantly better and faster than LJ, however it could not be determined conclusively which of the two media that provided the highest degree of decontamination when the incubation time was also included. SIGNIFICANCE AND IMPACT OF THE STUDY: HEYM should be the primary medium rather than LJ for detection of Map in cattle.     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Characterization of mesentericin ST99, a bacteriocin produced by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> subsp. <Emphasis Type="Italic">dextranicum</Emphasis> ST99 isolated from boza

Lactic acid bacteria isolated from Boza, a cereal-fermented beverage from Belogratchik, Bulgaria, were screened for the production of bacteriocins. With the first screening, 13 of the 52 isolates inhibited the growth of Listeria innocua and Lactobacillus plantarum. The cell-free supernatant of one of these strains, classified as Leuconostoc mesenteroides subsp. dextranicum ST99, inhibited the growth of Bacillus subtilis, Enterococcus faecalis, several Lactobacillus spp., Lactococcus lactis subsp. cremoris, Listeria innocua, Listeria monocytogenes, Pediococcus pentosaceus, Staphylococcus aureus and Streptococcus thermophilus. Clostridium spp., Carnobacterium spp., L. mesenteroides and Gram-negative bacteria were not inhibited. Maximum antimicrobial activity, i.e. 6,400 arbitrary units (AU)/ml, was recorded in MRS broth after 24 h at 30°C. Incubation in the presence of protease IV and pronase E resulted in loss of antimicrobial activity, confirming that growth inhibition was caused by a bacteriocin, designated here as mesentericin ST99. No loss in activity was recorded after treatment with -amylase, SDS, Tween 20, Tween 80, urea, Triton X-100, N-laurylsarcosin, EDTA and phenylmethylsulfonylfluoride. Mesentericin ST99 remained active after 30 min at 121°C and after 2 h of incubation at pH 2 to 12. Metabolically active cells of L. innocua treated with mesentericin ST99 did not undergo lysis. Mesentericin ST99 did not adhere to the cell surface of strain ST99. Precipitation with ammonium sulfate (70% saturation), followed by Sep-Pack C₁₈ chromatography and reverse-phase HPLC on a C₁₈ Nucleosil column yielded one antimicrobial peptide.