Bacillus cereus CH2对茄子黄萎病的田间防治效果研究以及对根围微生态群落结构的影响

拟对Bacillus cereus CH2菌株在田间条件下,对茄子黄萎病的防治效果及其对茄子根围土壤微生物群落结构和功能的影响进行研究。田间试验显示蜡状芽孢杆菌Bacillus cereus CH2对茄子黄萎病有60.6%的防治效果。对菌剂和清水对照处理小区茄子根围土壤的Biolog分析显示,CH2的使用不会对根围土壤原有微生物群落结构和功能多样性产生显著影响,同时对六大类碳源利用能力的分析结果显示,CH2的使用能在植株生长前期提高根围土壤微生物群体对除酚类化合物的五大类碳源的利用能力。     

Characterization of chitinase secreted by <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain CH2 and evaluation of its efficacy against Verticillium wilt of eggplant

A chitinase-secreting strain CH2 was one of 353 strains isolated from rhizosphere of eggplant. Based on 16S rDNA sequence alignment and several biochemical and physiological characteristics, the strain was identified to be of Bacillus cereus. On chitin–Ayers (CA) medium, the strain secreted chitinase. Evaluation of its activity, combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), showed it to be a 15.0-KD chitinase. On glass slides, germination of the fungal spores was effectively suppressed by the bacterial suspension, supernatant from the suspension, and 0.005% solution of chitinase extracted from the strain CH2. The optimum pH for chitinase was 7.1 and optimum temperature was 40°C. At that temperature, high-level chitinolytic activity was retained for 10 days. In greenhouse experiments, suspension of the cells of the CH2 strain reduced the severity of Verticillium wilt on eggplant by 69.69%, its supernatant by 54.04%, and the enzyme diluted to 0.01% strength by 53.13% in 14 days. Strain CH2 and its chitinase have good commercial potential in controlling Verticillium wilt.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.     

Suppression of plant wilt diseases by nonpathogenic Fusarium oxysporum Fo47 combined with actinomycete strains

In order to build integrated strains with superior growth-promoting and disease-suppression effects, the biological control efficacy of Fo47 solid agents combined with actinomycetes strains toward Fusarium oxysporum and Verticillium dahliae were investigated in experiments on watermelon, cotton and eggplant. Five actinomycetes strains were prepared by solid fermentation. The count of viable solid agents, initially with propagules at 10⁷–10¹¹ CFU/g, slowly decreased after being stored one year at room temperature. After being inoculated into sterile soil for 50 days, the viable count of strain Fo47 remained at a stable level. The suppressive effects of Fo47 combined with strain QLP12 on Fusarium wilt on watermelon and cotton, and Verticillium wilt on eggplant, reaching 58.47%, 50.73% and 58.82%, respectively. This was significantly better than the single strain Fo47 alone, and growth of these treated plants and the colonisation rate of Fo47 were increased substantially as well. These results indicate that solid integrated agents with a high viability count and superior stability in soil could increase disease suppression and promote plant growth by synergy with different strains. The increased suppression obtained by Fo47 combined with actinomycete strains was not due to a simple addition of different mechanisms of biocontrol agents. By being intelligently integrated, these combinations increase disease suppression and provide the best biocontrol effect.     

Use of Grafting and Calcium Cyanamide as Alternatives to Methyl Bromide Soil Fumigation and their Effects on Growth, Yield, Quality and Fusarium Wilt Control in Melon

Grafting melon (Cucumis melo L.) seedlings on to the Fusarium oxysporum f. sp. melonis (Fom) commercial resistant squash rootstocks ‘Mamouth’ and ‘Nun 9075 RT’ and soil sterilization with calcium cyanamide (CaCN₂, Perlka) were tested in 2001 and 2002 as alternatives to methyl bromide (MB) soil fumigation. Ungrafted seedlings of the F₁ melon hybrid ‘Galia’ were cultivated: (i) in soil sterilized by MB and then artificially infested with Fom (this served as a control), (ii) in soil artificially infested with Fom and then sterilized by MB, (iii) in soil artificially infested with Fom and then sterilized with CaCN₂ (Perlka), grafted seedlings on the commercial rootstocks, (iv) ‘Mamouth’ and (v) ‘Nun 9075 RT’ were cultivated in soil sterilized with MB and then artificially infested with Fom. The grafted plants on ‘Mamouth’ and ‘Nun 9075 RT’ and plants in the Perlka treatment (2001) developed mild symptoms, as indicated by the significantly lower leaf symptom index (LSI; average values 1.06, 1.08 and 1.07) and disease index (DI; average values 1.60, 1.25 and 2.33), respectively, when compared with the controls (average values of LSI = 2.65 and DI = 5.06). Plants grafted on ‘Mamouth’ and ‘Nun 9075 RT’ and plants in the Perlka treatment (2001) were more vigorous than the controls as assessed on plant height, stem diameter and root biomass. When compared with the controls, this resulted in an increased (over years) early production (326.3, 265.8 and 489.1%) and late production (371.0, 357.0 and 404.2%). Fruit size was also larger in early production (29.2, 50.9 and 32.3) and late production (4.3, 15.2 and 26.0). The total soluble solids (^oBrix) increased (over years) in early production (27.4, 39.6 and 47.9) and late production (7.59, 10.07 and 5.6) when compared with the controls. Thus, grafting on resistant squash rootstocks ‘Mamouth’ and ‘Nun 9075 RT’ and soil sterilization with Perlka had positive effects on growth, production and fusarium wilt control in melon.     

云南野生茄资源黄萎病苗期人工接种抗性鉴定分析

黄萎病是目前茄子生产中的主要病害之一,广泛收集、鉴定、筛选抗性资源,尤其是从野生近缘种中发掘抗性基因并培育抗病品种,是解决茄子黄萎病危害的优选途径。云南省拥有丰富的野生茄子资源但尚未有效利用。本研究针对云南省茄子主产区收集到的3种黄萎病菌株,通过形态学鉴定和真菌18S r DNA/ITS鉴定,均属于大丽轮枝菌,并通过致病力鉴定,筛选出一株强致病力菌株(QZ-S);应用菌株QZ-S,通过苗期人工接种的方法对45份云南野生茄子资源开展黄萎病抗性鉴定,最终筛选出2份高抗材料(蒜芥茄和喀西茄)、2份抗病材料(水茄和多裂水茄)、6份中抗材料(1份刺天茄和5份红茄);此外,还筛选到了1份黄萎病高感材料239-3-2。本研究筛选出的材料可应用于茄子黄萎病抗病育种,为茄子及其他作物黄萎病抗病育种提供抗源。     

Vegetative compatibility and pathogenecity of Verticillium dahliae kleb. isolates from olive in Iran

Verticillium wilt caused by Verticillium dahliae is a serious problem of olive trees leading to significant reduction in yield. Verticillium wilt of olive trees was first recorded in Iran 1996 and confirm as due to Verticillium dahliae Kleb. 101 isolates of V. dahliae from olive trees at deferent locations in north provinces of Iran were assigned to vegetative compatibility groups (VCGS), using nitrate non-utilizing (Nit) mutants. A higher frequency of nit 1/nit 3 mutants (93%) was obtained compared with NitM (7%) with 10% of the isolates being assigned to VCG1 and 51% VCG4B and 19% VCG2A. 20% of isolates could not be classified in standard isolates. The pathogenecity of 15 randomly selected isolates (5 of each VCG) was tested on olive seedling (cv. Zard) and eggplant. The VCGs isolates were similarly aggressive on olive. However, VCG1 isolates were more aggressive on eggplant cv. Local than the VCG2A and VCG4B isolates as indicated by a higher colonization index. The pathogenecity tests of the pathogen on test plants (cotton cv. 'sahel', eggplant cv. 'local' and tomato cv. 'ps') show all isolates category in 2 pathogenecity groups defoliate and non-defoliate (with severe and mild subgroups). The morphology of V. dahliae isolates on C'zapeck's agar and water agar medium were different especially for microsclerotia appearance time in culture and their morphology.     

Effects of Drip Irrigation on Population of Verticillium dahliae in Olive Orchards

Verticillium wilt of olive (Olea europaea L.), caused by Verticillium dahliae, is nowadays the most serious olive disease in Spain. The disease increments are being observed particularly in young olive plantations, favoured by several factors including inadequate cultural practices and crop production intensification, such as irrigation. Thus, three olive orchards affected by Verticillium wilt, with disease incidence ranging 30–50%, were selected to determine if the drip irrigation could favour the increase of pathogen in soil. Pathogen in soil was quantified in wet zones around the drippers and in dry zones out of them. Inoculum density in all experiments was higher in wet than in dry areas. After 4 months of watering, soil pathogen population increased considerably in wet and dry areas but inoculum density remained higher in the wet soil.     

Transfer of tomato immune receptor Ve1 confers Ave1‐dependent Verticillium resistance in tobacco and cotton

Verticillium wilts caused by soilborne fungal species of the Verticillium genus are economically important plant diseases that affect a wide range of host plants and are notoriously difficult to combat. Perception of pathogen(‐induced) ligands by plant immune receptors is a key component of plant innate immunity. In tomato, race‐specific resistance to Verticillium wilt is governed by the cell surface‐localized immune receptor Ve1 through recognition of the effector protein Ave1 that is secreted by race 1 strains of Verticillium spp. It was previously demonstrated that transgenic expression of tomato Ve1 in the model plant Arabidopsis thaliana leads to Verticillium wilt resistance. Here, we investigated whether tomato Ve1 can confer Verticillium resistance when expressed in the crop species tobacco (Nicotiana tabcum) and cotton (Gossypium hirsutum). We show that transgenic tobacco and cotton plants constitutively expressing tomato Ve1 exhibit enhanced resistance against Verticillium wilt in an Ave1‐dependent manner. Thus, we demonstrate that the functionality of tomato Ve1 in Verticillium wilt resistance through recognition of the Verticillium effector Ave1 is retained after transfer to tobacco and cotton, implying that the Ve1‐mediated immune signalling pathway is evolutionary conserved across these plant species. Moreover, our results suggest that transfer of tomato Ve1 across sexually incompatible plant species can be exploited in breeding programmes to engineer Verticillium wilt resistance.     

Inhibition of tomato shoot growth by over‐irrigation is linked to nitrogen deficiency and ethylene

Although physiological effects of acute flooding have been well studied, chronic effects of suboptimal soil aeration caused by over‐irrigation of containerized plants have not, despite its likely commercial significance. By automatically scheduling irrigation according to soil moisture thresholds, effects of over‐irrigation on soil properties (oxygen concentration, temperature and moisture), leaf growth, gas exchange, phytohormone [abscisic acid (ABA) and ethylene] relations and nutrient status of tomato (Solanum lycopersicum Mill. cv. Ailsa Craig) were studied. Over‐irrigation slowly increased soil moisture and decreased soil oxygen concentration by 4%. Soil temperature was approximately 1°C lower in the over‐irrigated substrate. Over‐irrigating tomato plants for 2 weeks significantly reduced shoot height (by 25%) and fresh weight and total leaf area (by 60–70%) compared with well‐drained plants. Over‐irrigation did not alter stomatal conductance, leaf water potential or foliar ABA concentrations, suggesting that growth inhibition was not hydraulically regulated or dependent on stomatal closure or changes in ABA. However, over‐irrigation significantly increased foliar ethylene emission. Ethylene seemed to inhibit growth, as the partially ethylene‐insensitive genotype Never ripe (Nr) was much less sensitive to over‐irrigation than the wild type. Over‐irrigation induced significant foliar nitrogen deficiency and daily supplementation of small volumes of 10 mM Ca(NO₃)₂ to over‐irrigated soil restored foliar nitrogen concentrations, ethylene emission and shoot fresh weight of over‐irrigated plants to control levels. Thus reduced nitrogen uptake plays an important role in inhibiting growth of over‐irrigated plants, in part by stimulating foliar ethylene emission.     

Increased resistance to fungal wilts in transgenic eggplant expressing alfalfa glucanase gene

The wilt diseases caused by Verticillium dahliae and Fusarium oxysporum are the major diseases of eggplant (Solanum melongena L.). In order to generate transgenic resistance against the wilt diseases, Agrobacterium-mediated gene transfer was performed to introduce alfalfa glucanase gene encoding an acidic glucanase into eggplant using neomycin phosphotransferase (npt-II) gene as a plant selection marker. The transgene integration into eggplant genome was confirmed by Polymerase chain reaction (PCR) and Southern blot analysis and transgene expression by the glucanase activity and western blot analysis. The selected transgenic lines were challenged with V. dahliae and F. oxysporum under in vitro and in vivo growth conditions, and transgenic lines showed enhanced resistance against the wilt-causing fungi with a delay of 5–7 days in the disease development as compared to wild-type plants.     

Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.     

The effect of reduced phytoalexin production on the resistance of upland cotton (Gossypium hirsutum) to verticillium and fusarium wilts

The effect on disease development of inhibiting the production of the sesquiterpenoid phytoalexin hemigossypol (HG) in cotton resistant to both verticillium and fusarium wilts was investigated. Inhibition was achieved by treating the plants with the sodium salt of compactin, a competitive inhibitor of hydroxy-methylglutaryl (HMG) CoA reductase. Compactin treatment (150 μg litre^-1) reduced HG production by a mean of 48%. The enzyme inhibitor did not mimic symptoms in uninfected plants or significantly reduce the ability of the conidia of either Fusarium oxysporum f.sp. vasinfectum or Verticillium dahliae to germinate. Treatment of infected plants with compactin resulted in a breakdown of resistance to verticillium wilt but not to fusarium wilt. These results support the view that HG production is the primary mechanism of resistance to verticillium wilt, but not to fusarium wilt.     

Seedling vaccination by stem injecting a conidial suspension of F2, a non-pathogenic Fusarium oxysporum strain, suppresses Verticillium wilt of eggplant

Several bacterial and fungal strains have been evaluated as biocontrol agents (BCAs) against Verticillium dahliae. In these studies, the BCAs were applied as a root drenching inoculum; however, this application method may have an adverse effect on the native, beneficial for the plants, microbial community. In the present study, it was evaluated whether endophytic application by stem injecting a conidial suspension of the non pathogenic Fusarium oxysporum F2 strain, isolated from a V. dahliae suppressive compost amendment, could reduce significantly Verticillium wilt symptom development in eggplants. It was revealed that stem injection of F2 seven days before transplanting the seedlings to soil infested by V. dahliae microsclerotia resulted in reduced disease severity compared to the control treatment. To visualise F2 ramification into the plant vascular system eggplant stems were injected with an EGFP transformed F2 strain. It was shown that F2 colonises effectively the plant vascular tissues over a long period of time as it was assessed by F2 DNA levels. In parallel, qPCR analysis showed that the application of F2 reduced significantly the amount of V. dahliae DNA in the stem tissues compared to the control treatment.     

Soil Salinity and Water Stress and Their Effect on Susceptibility to Verticillium Wilt Disease, Ion Composition and Growth of Pistachio

The effects of soil salinity and water stress on Verticillium wilt, ion composition and growth of pistachio were studied in a greenhouse experiment (18–32°C). Treatments consisted of three levels of salinity (0, 1200 and 2400 mg NaCl/kg soil), three levels of water stress (3, 7 and 14 day irrigation regimes) and two Pistachio cultivars (Sarakhs and Qazvini, common rootstocks in Iran). Infested soil containing 50 microsclerotia/g of a pistachio isolate of Verticillium dahliae was used for all treatments and non‐infested soils were used as control. The experiment was arranged in a completely randomized design with three replications. Eight‐week‐old pistachio seedlings were transferred to infested and non‐infested soil and then exposed to salt stress and thereafter water stress. Shoot dry weights of both rootstocks were reduced significantly with increasing NaCl levels; however, increasing irrigation regimes reduced salt injury. Salt stress significantly increased shoot and root colonization by V. dahliae in both cultivars. Moreover, increasing of salinity level was positively correlated with increasing concentrations of Na⁺, K⁺ and Cl^? in both cultivars, but negatively correlated with increasing irrigation regimes. Based on these results, Sarakhs and Qazvini were found to be sensitive and tolerant to the effect of irrigation regimes, salinity and Verticillium wilt disease, respectively. Although there were no interactive effects of irrigation and salinity on V. dahliae infection.     

Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase

Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.     

The interactions of potato cyst nematodes and Verticillium dahliae on early and maincrop potato cultivars

The effects of potato cyst nematodes (Globodera rostochiensis and G. pallida) on the growth of early and later maturing potato cultivars, with or without the H, resistance gene, were compared in pot experiments and the effects of introducing Verticillium dahliae into the system were studied. The influence of the nematodes on root function and structural integrity was inferred from their effects on calcium and water uptake. Early maturing cultivars were less tolerant of nematode attack than later maturing ones, and those with the H₁ resistance gene were more tolerant of G. rostochiensis than G. pallida. When V. dahliae was introduced, Verticillium wilt symptoms appeared much earlier on the early than the maincrop cultivars. Also, Maris Anchor (first early) died before Maris Peer (second early) and death of both when infected by nematodes and fungus was much earlier than uninfected control plants or plants infected with G. rostochiensis only. Maris Peer infected by both organisms snowed symptoms much more severe than when infected by either organism alone, whereas Maris Anchor was almost as severely affected by V. dahliae alone as both organisms together. Pentland Javelin (first early) developed Verticillium wilt symptoms several weeks later than other cultivars and the addition of nematodes failed to increase the severity of the symptoms. These differences in cultivar susceptibility to the combination of nematodes and fungus may be due to the nematodes causing different degrees of trauma in the roots of the three cultivars: measuring calcium uptake per unit water uptake showed that nematodes dramatically increase calcium uptake but that the proportional change is least in Pentland Javelin. If this is a reflection of less damage by nematodes to the endodermis of this cultivar, it may explain the greater resistance of its roots to penetration by V. dahliae.     

The growth characteristics and yield potential of rice (Oryza sativa) under non‐flooded irrigation in arid region

The objectives of this field experiment were to study the growth characteristics and yield potential of rice plants under non‐flooded irrigation in arid area. Non‐flooded treatments included drip irrigation with plastic mulching treatments (DIs), furrow irrigation with plastic mulching treatment (FIM) and furrow irrigation with non‐mulching treatment (FIN). Conventional flooded cultivation (F) was check treatment (CK). The four drip irrigation treatments differed in the amount of water applied before and after panicle initiation. Root length density, leaf dry weight, shoot dry weight and root activity were generally higher in the non‐flood‐irrigated treatments (especially the drip‐irrigated treatments) than in the flood‐irrigated treatment at mid‐tillering. However, the growth and development of rice plants were limited after jointing in the non‐flooded irrigation treatments. Increasing the root/shoot ratio and root length density in the 20–40 cm depth and decreasing specific root length at 0–20 cm soil layer were important mechanisms for helping the rice plants to adapt to the non‐flooded environmental stresses. Finally, the grain yield in the non‐flooded irrigation treatments was lower than that in the F treatment. These low yields were mainly attributed to the low root length density at 0–20 cm depth and root activity. Generally speaking, the restricted degrees in the DIs were smaller than that in the FIM and FIN treatments. Among the DIs, both the highest grain yield (8223–8900 kg ha^?1) and the highest water use efficiency (WUE) (0.63) were observed when the soil water content was kept at ?30 kPa before panicle initiation and at ?15 kPa after panicle initiation (referred to as the DI2 treatment). The yield in the DI2 treatment was not significantly different than that in the flood‐irrigated treatment. However, WUE was 2.5 times higher in the DI2 treatment than in the F treatment. These results suggest that drip irrigation technology can be considered as a better water‐saving cultivation of rice plants in arid region.     

Effect of AMF application on growth, productivity and susceptibility to Verticillium wilt of olives grown under desert conditions

The effect of arbuscular mycorrhizal fungi (AMF) on olive (Olea europaea) growth and development was followed for 4 years after transplanting in irrigated commercial orchards under arid conditions. Sites I and II were irrigated with saline water (EC?=?4.5 dS/m). In site I, the soil was infested with Verticillium dahliae and olive varieties ‘Picual’ (Verticillium susceptible) and ‘Barnea’ (relatively Verticillium tolerant) were tested. In site II, the soil was virgin soil (previously non-cultivated soil) and olive varieties ‘Souri’ and ‘Barnea’ were tested. Plants for all sites were inoculated in the nursery with Glomus intraradices alone or in a mixture with G. mosseae. Relative to non-inoculated trees, AMF colonization enhanced vegetative growth, expressed as tree height and trunk circumference, at all sites. At first commercial harvest, AMF-treated trees had higher fruit and oil yields than non-mycorrhitic controls. Under saline water irrigation, differences between inoculated and non-inoculated treatments were reduced in the slow-growing ‘Souri’ but remained apparent in the modern fast-growing ‘Barnea’. AMF colonization did not appear to improve tolerance of either ‘Picual’ or ‘Barnea’ to V. dahliae, and both were more susceptible than the non-inoculated controls. Thus inoculation of olive plants with AMF improves transplant growth and adaptation in arid areas during the first 3 years of growth and until the first commercial harvesting season.