Isolation of Bacillus pumilus from in vitro grapes as a long-term alcohol-surviving and rhizogenesis inducing covert endophyte

AIMS: To isolate and characterize the alcohol-surviving covert bacterium associated with grape tissue culture. METHODS AND RESULTS: Single colony was isolated by plating the spent rectified spirit used during grape culturing and the organism was identified as Bacillus pumilus using partial 16S rDNA sequence data. Spotting tests (1 microl) using 3-day-old broth culture having a spore content of 20-30% showed similar bacterial survival in 25-90% (v/v) aqueous ethanol for 14 days. Survival in 90% ethanol and 90% rectified spirit appeared affected thereafter with no colony growth from 1 microl samples after 4 months. Plating the samples at this stage gave similar CFU ml(-1) for 25, 50 and 70% ethanol, a significant reduction in 80% ethanol and very few colonies in 90% ethanol and rectified spirit. B. pumilus-inoculated grape microcuttings showed substantial endophytic colonization of original cuttings (7.4 x 10(6) CFU g(-1)) followed by the sprout (5.9 x 10(5)) and roots (2.0 x 10(4)). The bacterium although a poor root colonizer, induced early rooting and more roots in vitro. Inoculation at ex vitro planting resulted in significantly more roots, root weight and shoot growth. CONCLUSIONS: Bacillus pumilus could remain as a covert endophyte in grape tissue cultures and survive in aqueous ethanol for extended periods. 90% ethanol was the most effective bactericidal concentration. The bacterium showed endophytic colonization and root and shoot growth promotion. SIGNIFICANCE AND IMPACT OF THE STUDY: The revelation that general recommendation of 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria, elucidation of the possibility of covert bacterial survival in vitro plant cultures and isolation of a potential plant growth promoting endophyte in grape.     

Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization

This study reports the effects of light availability during the acclimatization phase on photosynthetic characteristics of micropropagated plantlets of grapevine (Vitis vinifera L.) and of a chestnut hybrid (Castanea sativa × C. crenata). The plantlets were acclimatized for 4 weeks (grapevine) or 6 weeks (chestnut), under two irradiance treatments, 150 and 300 mol m^–2 s^–1 after in vitro phases at 50 mol m^–2 s^–1. For both treatments and both species, leaves formed during acclimatization (so-called `new leaves') showed higher photosynthetic capacity than the leaves formed in vitro either under heterotrophic or during acclimatization (so-called `persistent leaves'), although lower than leaves of young potted plants (so-called `greenhouse leaves'). In grapevine, unlike chestnut, net photosynthesis and biomass production increased significantly with increased light availability. Several parameters associated with chlorophyll a fluorescence indicated photoinhibition symptoms in chestnut leaves growing at 300 mol m^–2 s^–1. The results taken as a whole suggest that 300 mol m^–2 s^–1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.     

Grapes, galls, and geography: the distribution of nuclear and mitochondrial DNA variation across host-plant species and regions in a specialist herbivore

Studies of patterns of molecular variation in natural populations can provide important insights into a number of evolutionary problems. Among these, the question of whether geographic factors are more important than ecological factors in promoting population differentiation and ultimately speciation has been an important and contentious area in evolutionary biology. Systems involving herbivorous insects have played a leading role in this discussion. This study examined the distribution of molecular variation in a highly specialized gall-forming insect, grape phylloxera (Daktulosphaira vitifoliae Fitch), that is found on both sympatric and allopatric host-plant species of the genus Vitis. In addition, the relationship of insects in the introduced range in the United States to ancestral populations in the native range was examined. Evidence for differentiation along host-plant lines from both nuclear (RAPD) and mitochondrial (COI) DNA was confounded with the effect of geography. Differentiation was found where hosts were allopatric or parapatric, but no evidence was found for such differentiation on two hosts, V. vulpina and V. aestivalis, that are broadly sympatric. The question of population differentiation onto these sympatric hosts can be considered to be resolved--it has not occurred in spite of a long history of association. Evidence was equivocal, but suggestive of a period of divergence in allopatry prior to reestablishment of contact, for insects associated with another host plant species, V. cinerea, found in both sympatric and parapatric populations. A low level of diversity and placement of samples collected from the grape species V. riparia at the tip of a phylogenetic tree supports the hypothesis that this host has been recently colonized from populations from the Mississippi Valley. A polyphyletic origin for biotype B grape phylloxera was supported: Although most samples collected from vineyards in the introduced range in California had similar haplotypes, they were closely related to natives on V. vulpina from the Atlantic Coast-Piedmont region. All samples collected from vineyards in Oregon and Washington were closely related to natives on V. riparia in the northern United States.     

Status and future of disease protection and grape berry quality alteration by micro‐organisms in viticulture

Grapevine is one of the most widely grown fruit crops in the world. At present, however, there is much concern regarding chemical pollution in viticulture due to the application of chemical fungicides and fertilizers. One viticultural practice to resolve this issue is the application of micro‐organisms to grapevine as a substitute for chemicals. Some micro‐organisms act as an enhancer of grape berry quality as well as a suppresser of disease in grapevine through their antagonistic ability and/or systemic resistance inducing ability. Herein, we review current and prospective applications of micro‐organisms in viticulture.

Significance and Impact of the Study

In this review, we evaluate the applicability of micro‐organisms in viticulture. Micro‐organisms can improve grape berry quality through grapevine disease protection and grape berry quality alteration. Because the use of micro‐organisms to protect grapevine from plant diseases is safer than the use of chemical fungicides, the use of biofungicides in viticulture is expected to be enhanced by the increasing consumer concern towards chemical fungicides. Micro‐organisms also modify plant secondary metabolites for use as flavours, pharmaceuticals and food additives. Studies of micro‐organisms that promote polyphenol, anthocyanin and aroma compound biosynthesis are in progress with an eye to improving grape berry quality.     

Nocturnal and daytime stomatal conductance respond to root-zone temperature in ‘Shiraz’ grapevines

Background and Aims

Daytime root-zone temperature may be a significant factor regulating water flux through plants. Water flux can also occur during the night but nocturnal stomatal response to environmental drivers such as root-zone temperature remains largely unknown.

Methods

Here nocturnal and daytime leaf gas exchange was quantified in ‘Shiraz’ grapevines (Vitis vinifera) exposed to three root-zone temperatures from budburst to fruit-set, for a total of 8 weeks in spring.

Key Results

Despite lower stomatal density, night-time stomatal conductance and transpiration rates were greater for plants grown in warm root-zones. Elevated root-zone temperature resulted in higher daytime stomatal conductance, transpiration and net assimilation rates across a range of leaf-to-air vapour pressure deficits, air temperatures and light levels. Intrinsic water-use efficiency was, however, lowest in those plants with warm root-zones. CO₂ response curves of foliar gas exchange indicated that the maximum rate of electron transport and the maximum rate of Rubisco activity did not differ between the root-zone treatments, and therefore it was likely that the lower photosynthesis in cool root-zones was predominantly the result of a stomatal limitation. One week after discontinuation of the temperature treatments, gas exchange was similar between the plants, indicating a reversible physiological response to soil temperature.

Conclusions

In this anisohydric grapevine variety both night-time and daytime stomatal conductance were responsive to root-zone temperature. Because nocturnal transpiration has implications for overall plant water status, predictive climate change models using stomatal conductance will need to factor in this root-zone variable.     

First record of Fusarium vascular wilt on grapevine in Egypt

During the summer season of 2003 and 2004, wilt syndromes of grapevine leaves (Cv. crimson) and vascular discolouration of roots have been observed in 2-year-old grapevine plants in the field at two sides in Gharbeia Governorate, Egypt. First, symptoms of wilt began on bottom leaves borderline as chlorosis and then these turned to necrotic spots and the leaves died. Wilt symptoms were spread to apical associated with vascular discolouration of roots and stem basal. Routine isolations of discoloured root tissue from diseased plant yielded eight isolates of Fusarium oxysporum Schlechtend only where no other fungi were developed. Microscopic examination revealed the presence of three shapes of microconidia, first is avoid shape non-septate measuring 2.5–3.0 μm × 6–10 μm, second is cylindrical with one septa measuring 2.6 μm × 17.0 μm and third shape also cylindrical with two septate measuring 3.0 μm × 20.0 μm. Macroconidia was rarely with three septate measuring 3.5– 4.0 μm × 35.0–38.0 μm, and chlamydospores were found singly or in pairs or chains. F. oxysporum isolates attacked grapevine plants (Cv. crimson) causing vascular wilt (66.7%) and root-rot syndrome (33.3%). In vitro isolates of F. oxysporum causing wilt of grapevine (Cv. crimson) varied for producing lytic enzymes, i.e. polygalacturonase (PG) and cellulase. The reactions of several grapevines (Cvs.) with a virulent isolate of F. oxysporum indicated the presence of two different symptoms, i.e. vascular wilt only on grapevine plants (Cv. crimson) and root-rot on the other grapevine (Cvs.), i.e. superior, Thompson, King robi and flame seedless. All F. oxysporum isolates caused vascular wilt of grapevine Cv. crimson, successfully reisolated from symptomatic vascular infected tissue and complete identification on the basis of colony, conidia morphology and host range at formae speciales level as F. oxysporum f. sp. herbemontis (Tochetto) Gordan. This is the first report of Fusarium wilt on grapevine in Egypt.     

四个酿酒葡萄品种组培快繁体系的初建

为了提高酿酒葡（Vitis vinifera）苗木繁殖速度及苗木品质,以‘赤霞珠’、‘西拉’、‘霞多丽’和‘美乐’4／p品种为试材,研究无菌外植体建立、启动培养、增殖培养和驯化移栽环节的关键技术,初步建立酿酒葡萄组培快繁体系。结果表明,以半木质化茎段为外植体接种成活率高,在培养基MS＋IBA0．2mg·L-1＋6．BA1．0mg·L-1＋KT0．5mg·L-1上启动培养外植体单芽萌发率最高,以培养基1／2MS＋IBA0．2mg·L-1＋KT1．0mg·L-1增殖培养兼生根诱导,组培苗生长健壮,繁殖率高。增殖培养6代后,‘赤霞珠’、‘西拉’、‘霞多丽’和‘美乐’分别由12株葡萄苗扩繁为l383株、1095株、744株和100株。组培苗驯化培养3周后移栽至营养钵,4个品种成活率均在72％以上。此组培快繁体系基本适用于4个酿酒葡萄品种,可应用于科学研究及工业化大规模生产。     

葡萄胚胎发育与败育过程中胚珠的多胺含量变化

花后40~60d期间,种子败育型无核葡萄胚珠的3种多胺含量明显低于有核葡萄,据此认为无核葡萄胚珠中多胺含量急剧下降和较低的多胺水平可能是导致葡萄胚胎败育的重要原因。(Spd Spm)/Put和Spm/PAs比值变化与葡萄胚胎发育密切相关,较低的比值不利于胚胎的正常分化。