Persistence of GR7 and Striga germination stimulant(s) from Euphorbia aegyptiaca Boiss. in soils and in solutions

The activity of GR7 and Striga germination stimulant(s) from Euphorbia aegyptiaca Boiss. showed adequate persistence (6–8 days) in acidic soils (pH 5·0–6·3), but residual activity was short (1–3 days) in alkaline soils. The compounds tended to lose activity at a faster rate in the alkaline clay Gezira soil (pH 7·8), than in its sandy equivalent (pH 8·1). In solution, pH had no influence on initial activity, but residual action was reduced more rapidly by alkalinity and high temperature. However, the rate of loss of activity in solution was slower than in soils.     

Modulation of host pH during the wheat–Fusarium culmorum interaction and its influence on the production and activity of pectolytic enzymes

The effect on ambient pH of Fusarium culmorum during its growth on mineral medium and in apoplastic fluids from infected wheat seedlings, and the effect on the production and activity of the enzymes pectin lyase (PNL) and polygalacturonase (PG), were investigated. Fungal development on a weakly buffered mineral medium in the pH range 5·0–8·0, with pectin as the sole carbon source, led to pronounced alkalinization, reaching values above 8·0. The increase in ambient pH was accompanied by enhancement of total PNL activity. Pectin lyase secretion was detected at pH 5·0 as a single isoenzyme. An additional isoenzyme was apparent during the increase in medium pH. Polygalacturonase was detected as a single isoenzyme only during early growth on medium buffered at pH 5·0. At this stage, the initial medium pH of 5·0, corresponding to the optimum pH for PG activity, appeared to be the most suitable for the activation of early production of this enzyme. During growth in acidified yeast extract medium the fungus secreted ammonia and increased medium pH. Similarly, in apoplastic fluids from inoculated seedlings the concomitant ammonia accumulation and rise in pH were recorded. This trend was accompanied by an increase in PNL, which could therefore function at close to its optimal pH. The results suggest that during infection of wheat seedlings by F. culmorum , pH modulation can lead to PNL production and activity, thus promoting colonization of host tissue.     

Efficacy of some rhizospheric and endophytic bacteria <Emphasis Type="Italic">in vitro</Emphasis> and as seed coating for the control of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> infecting durum wheat in Tunisia

Sixty two rhizospheric and endophytic bacterial strains were evaluated for their biocontrol effect on two aggressive Fusarium culmorum isolates (Fc2 and Fc3). We observed that 35 % and 23 % of the tested strains inhibited the in vitro growth of Fc2 and Fc3 respectively. The observed antagonism was due to inhibition by contact (13–19 % of the strains) or at distance (10–16 % of the strains) for both fungal isolates. Some of the antagonistic bacteria showed the ability to produce diffuse and/or volatile compounds that inhibit the growth, the sporulation and macroconidia germination of F. culmorum. None of the tested antagonistic bacteria showed chitinase activity on synthetic medium. The sequencing of the 16S rDNA genes of some antagonistic bacteria showed that they belong to the genera Bacillus, Pseudomonas and Microbacterium. The double inoculation of durum wheat seeds by the antagonistic bacterial strains (B13, B18, BSE1, BSE3 and B16E) and the two F. culmorum isolates showed that germination and seedling vigor were generally improved in vitro. The percentage of infected seeds was also reduced. In greenhouse trials, the biocontrol effectiveness of F. culmorum was dependant from the virulence of the fungal strain and the specificity of the antagonistic interaction between bacterial and fungal strains. The bacterial strains B18 and B16E reduced F. culmorum infection on durum wheat plants probably due to their antagonistic and plant growth promoting activities and they may be used in a mixture as seed biopriming inoculum for plant growth bio-promoting and Fusarium wheat diseases biocontrol.     

Nematicidal activity of the leaf powder and extracts of Myrtus communis against the root‐knot nematode Meloidogyne javanica

Nematicidal activity of the leaf powder, leaf extracts and formulated leaf extracts of Myrtus communis, an evergreen shrub that is widely distributed in Israel and other Mediterranean countries, was evaluated using the root‐knot nematode Meloidogyne javanica in in vitro and pot experiments. Leaf powder added to sand at 0·1% (w/w) reduced the number of juveniles recovered from the sand by more than 50%. Reduction in galling index and number of nematode eggs on tomato roots was also observed by incorporating the leaf powder at 0·1–0·4% (w/w) in the soil in pot experiments. Leaf powder extracts with methanol or ethanol showed the highest nematicidal activity among all extracts tested. Emulsifiable concentrates of leaf‐paste extract at a concentration as low as 0·005% (a.i., w/w) reduced the number of juveniles recovered from treated sand and the gall index of cucumber seedlings. The extract paste at 26 g m^?2 was also effective in reducing the gall index of tomato plants in field‐plot experiments. The leaf powder at 0·2% and the formulated leaf‐paste extract at 0·02% were also nematicidal to Tylenchulus semipenetrans and Ditylenchus dipsaci, but not to Pratylenchus penetrans or Steinernema feltiae. At least three nematicidal compounds were found in the leaf extract upon fractionation by thin‐layer chromatography. The results suggest that the leaf powder and paste extract of M. communis are potential nematicides against root‐knot nematodes.     

Correlation of in planta endo‐beta‐1,4‐xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola

The association of the cell wall degrading enzyme endo‐beta‐1,4‐xylanase (EC 3.2.1.8) with pathogenicity of Mycosphaerella graminicola was examined in planta. The enzyme production of two M. graminicola isolates (T0372 and T0491), as well as their ability to infect seedlings of susceptible wheat cv. Scorpion, was first compared. No significant difference was found between the two isolates regarding spore germination rates, mycelial growth on the leaf surface or direct and stomatal penetrations. However, restricted hyphal growth was observed inside leaves inoculated with T0372, whereas successful mesophyll colonization with a strong intercellular fungal growth was found in leaves infected with T0491. Likewise, T0372 was unable to induce lesions bearing pycnidia and to produce endo‐beta‐1,4‐xylanase activity until 22 days post‐inoculation (d.p.i.). On the other hand, significant high increases of both diseased leaf area bearing pycnidia and endo‐beta‐1,4‐xylanase activity were observed between 16 and 22 d.p.i. for T0491 (r = 0·98). The investigation of 24 additional isolates, including the IPO323 and IPO94269 reference isolates, highlighted a strong correlation between endo‐beta‐1,4‐xylanase activity and disease development levels (r = 0·94). This study demonstrates that differences in pathogenicity in M. graminicola are not linked to events on the leaf surface or to frequency of leaf penetration, but to the ability of the fungus to colonize the mesophyll and to produce the cell wall degrading enzyme endo‐1,4‐beta‐xylanase during the necrotrophic phase.     

Transmission of ‘Candidatus Liberibacter solanacearum’ in carrot seeds

A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.     

Colonization of soft wheat following infection of the stem base by Fusarium culmorum and translocation of deoxynivalenol to the head

Following inoculation of the base of soft wheat seedlings with Fusarium culmorum, disease symptoms typical of crown rot developed at the stem base and extended up to the third node by plant maturity. Fungus was isolated from all tissues exhibiting symptoms but not from symptomless tissues. Histopathological analysis revealed that the fungus was present mainly in the parenchymatic cells of the stem base and colonized the tissues via apoplastic and symplastic pathways. Host response in advance of pathogen colonization was observed. At maturity, plants were divided into sections from the inoculated area to the head. Heads were also separated into grain, rachis and chaff components. Colonization by the fungus was assessed by isolation from surface‐sterilized segments and quantified by real‐time PCR. Disease symptoms and the fungus were detected up to the third node, while deoxynivalenol (DON) was present in all stem segments and heads. Within the head, the DON concentration was higher in the rachis than in the chaff and grain components. These results demonstrate that F. culmorum can extensively colonize stem tissues but not reach the head by the time of plant maturity. In contrast, DON was detected in tissues beyond those colonized by the fungus, translocating to the head where, although accumulating mainly in the rachis, significant quantities accumulated in the grain. These findings indicate that there is a potential threat of contamination of grain with DON where severe crown rot is present in a crop.     

Evaluation of different methods for the characterization of carrot resistance to the alternaria leaf blight pathogen (Alternaria dauci) revealed two qualitatively different resistances

Alternaria leaf blight (ALB), caused by Alternaria dauci, is one of the most damaging foliar diseases of carrot worldwide. The aim of this study was to compare different methods for evaluating levels of carrot resistance to ALB. Three techniques were investigated by comparison with a visual disease assessment control: in vivo conidial germination, a bioassay based on a drop‐inoculation method, and in planta quantification of fungal biomass by quantitative PCR (Q‐PCR). Three carrot cultivars showing different degrees of resistance to A. dauci were used, i.e. a susceptible cultivar (Presto) and two partially resistant genotypes (Texto and Bolero), challenged with an aggressive or a very aggressive isolate of A. dauci. Both partially resistant genotypes produced a higher mean number of germ tubes per conidium (up to 3·42±0·35) than the susceptible one (1·26±0·18). The drop‐inoculation results allowed one of the partially resistant genotypes (Bolero, log₁₀(S+1) = 1·34±0·13) to be distinguished from the susceptible one (1·90±0·13). By contrast, fungal growth measured by Q‐PCR clearly differentiated the two partially resistant genotypes with log₁₀(I) values of 2·77±0·13 compared to the susceptible cultivar (3·65±0·13) at 15 days post‐inoculation. This result was strongly correlated (r² = 0·91) with the disease severity index scored at the same date. Data obtained with the different assessment methods strongly suggest that the Texto and Bolero genotypes have different genetic resistance sources.     

Callose and β‐1,3‐glucanase inhibit Phytophthora cinnamomi in a resistant avocado rootstock

Phytophthora root rot (PRR) of avocado, caused by Phytophthora cinnamomi, is a significant threat to sustainable production wherever the crop is grown. Resistant rootstocks in combination with phosphite applications are the most effective options for managing this disease. Recently, the mechanisms underpinning PRR resistance have been investigated by the avocado community. Here, biochemical assays and confocal and scanning electron microscopy were used to investigate early defence responses in PRR resistant and ‐susceptible avocado rootstocks. Zoospore germination and subsequent hyphal growth for the pathogen were significantly inhibited on the surface of resistant avocado roots. When penetration occurred in the resistant R0.06 rootstock, callose was deposited in the epidermal cells, parenchyma and cortex of roots. In addition, β‐1,3‐glucanase was released early (6 h post‐inoculation, hpi) in response to the pathogen, followed by a significant increase in catalase by 24 hpi. In contrast, susceptible R0.12 roots responded only with the deposition of lignin and phenolic compounds incapable of impeding pathogen colonization. In this study, PRR resistance was attributed to a timely multilayered response to infection by P. cinnamomi.     

螺旋毛壳ND35 β-1,3-葡聚糖酶的诱导、性质及其抑菌作用

 以病原菌Rhizoctonia solani的细胞壁为诱导物,模拟毛壳菌自然的重寄生过程,研究了内生真菌螺旋毛壳(Chaetomium spirale) ND35 β-1,3-葡聚糖酶的产酶条件、性质,尤其是不同碳源的调控作用。结果表明,不同种类的真菌细胞壁及几丁质和昆布多糖,均可诱导产生β-1,3-葡聚糖酶,而作为分解代谢产物的葡萄糖则抑制产酶。经硫酸铵沉淀、DEAE-Sepharose阴离子交换层析及Phenyl-Sepharose疏水层析,并通过SDS-PAGE鉴定,纯化了一种分子量约为73 kDa的内切β-1,3-葡聚糖酶GLUC73。其最适反应温度为55℃,在40℃以下较稳定;最适pH值为5.5,在pH 5-9范围内均很稳定;酶活性受Hg²⁺、Fe³⁺、Zn²⁺、Mg²⁺等金属离子不同程度的抑制,Mn²⁺和Co²⁺对酶有激活作用;以昆布多糖为底物时,该酶的米氏常数K_m为0.412 mg·mL^-1,最大反直速度V_max为3.876 U·mL^-1。粗酶液同时具有β-1,3-葡聚糖酶和几丁质酶活性,离体抑菌试验表明,对苹果炭疽病菌(Glomerella cingulata)、杨树腐烂病菌(Valsa sordida)、苹果树腐烂病菌(Valsa mali)的菌丝生长和孢子萌发有明显的抑制作用。通过对β-1,3-葡聚糖进行免疫细胞化学标记和超微结构观察,间接证明了β-1,3-葡聚糖酶在螺旋毛壳重寄生过程中的作用。     

The Relationship Between Wheat Seed Weight, Infection by Fusarium culmorum or Microdochium nivale, Germination and Seedling Disease

The distribution of seeds by weight for three lots of winter wheat cv. Avalon infected by Fusarium culmorum and three lots of winter wheat cv. Riband infected by Microdochium nivale was determined. The distribution of infected seeds within each seed lot was then determined by isolating F. culmorum from seeds on moist filter paper and M. nivale from seeds on potato dextrose agar. The distribution of M. nivale infected seeds between seeds of different weight was similar to that of the seed lot as a whole, whereas the distribution of F. culmorum was greater in light seeds than heavy seeds. The percentage germination of infected seeds decreased with seed weight. A similar situation was found with respect to seedling emergence in compost for F. culmorum infected seeds. However, with M. nivale infection, similar numbers of seedlings emerged from both light and heavy infected seeds. Seed treatment with guazatine increased seedling emergence for both light and heavy seed infected by M. nivale. However, seedling emergence from F. culmorum infected seed was poor even following treatment with guazatine. Poor emergence was most evident from light seed.     

Yucca schidigera extract: a potential biofungicide against seedborne pathogens of sorghum

Treatment of seeds with an aqueous extract of yucca (YE), Yucca schidigera, was evaluated for antifungal activity against seedborne pathogens as well as its effect on seed germination and seedling growth of sorghum (Sorghum bicolor). The antifungal effect of YE was observed against Leptosphaeria sacchari (syn. Phoma sorghina) when the extract was applied at 2·5 and 10% concentrations. At 10% concentration, YE significantly reduced not only the incidence of L. sacchari, but also that of Fusarium spp., Cochliobolus lunatus (syn. Curvularia lunata) and Cladosporium spp. The effect of 10% YE on seedborne fungi was broader than the fungicide fludioxonil, particularly with regard to Fusarium. Furthermore, the number of normal, healthy‐looking seedlings increased in a dose‐responsive manner with YE treatment. Seedling vigour was also stimulated by YE but no correlation was observed with the concentrations tested. Under glasshouse conditions, the treatment of seeds with 10% YE increased the emergence of seedlings and plant height and reduced the number of seedlings with crown rot compared to negative controls and saponin. The positive effect was similar to the effect obtained with fungicide‐treated seeds. Treatment of seeds with synthetic saponin inhibited seedborne fungi less effectively and also negatively affected germination and vigour of the seedlings, compared to the treatment with YE. The results demonstrate an agronomic potential for the use of YE as a biofungicide for seed treatment of sorghum. The difference between the antifungal and the vigour‐stimulating effects of YE warrants further investigation.     

Accumulation of pathogenesis-related proteins in the epidermis of tomato leaves infected by Cladosporium fulvum

Upon infection byCladosporium fulvum, tomato plants start to produce pathogenesis-related (PR) proteins. The PR proteins 1,3-β-glucanase, chitinase, and PR-1b accumulated near the stomata in the lower epidermis ofC. fulvum-inoculated tomato leaves as could be determined by immunolocalization with polyclonal antibodies. However, no differences in accumulation of PR proteins between a compatible and an incompatible interaction were found. Results obtained from enzyme activity measurements of 1,3-β-glucanase and chitinase on similar leaf material as used for the immunolocalization did not fully reflect the immunolocalization data. The antibodies possibly detect only the extracellular but not the intracellular enzymes. The accumulation of PR proteins near the stomata might be part of a general defence response of plants against pathogens and potential pathogens.     

Adjustment of soil-surface pH and comparison with conventional fungicide treatments for control of lettuce drop (Sclerotinia minor)

The use of soil-surface applications of finely powdered calcium hydroxide (Ca(OH)₂) to inhibit Sclerotinia minor sclerotial germination and infection at the collar region of lettuce plants is described. In the laboratory, a pH > 8·0 reduced sclerotial germination of the three S. minor isolates tested. In the glasshouse, surface applications of 2–10 t Ca(OH)₂ ha⁻¹ raised the pH of the top 1–2 cm of a duplex sandy loam soil above 8·5 for at least 8 weeks without affecting soil pH within the transplant root zone. There was a linear relationship between the rate of Ca(OH)₂ applied and disease control, with complete disease suppression at 10 t Ca(OH)₂ ha⁻¹. In field trials on two soil types (duplex sandy loam, pH 6·0; and red ferrosol, pH 6·9), a rate of 2·5 t Ca(OH)₂ ha⁻¹, maintained soil-surface pH above 8·5 for 1–3 weeks and provided up to 58% reduction in lettuce drop. Application of polyvinyl alcohol (a soil-conditioning polymer) over the Ca(OH)₂ layer appeared to reduce Ca(OH)₂ loss by wind, but did not improve retention of raised soil-surface pH or disease suppression. Ca(OH)₂ treatment gave similar disease control to the industry standard treatment of a procymidone-based fungicide seedling drench. A combined treatment of Ca(OH)₂ and fungicide drench gave greater control than either individual treatment, and equivalent control to fungicide drench and three procymidone foliar sprays, offering integrated management options. The use of soil-surface-applied Ca(OH)₂ with fungicides, rotation and drip irrigation offers an opportunity for enhanced and sustainable control of lettuce drop.     

Factors affecting seed germination and emergence of Gomphrena perennis

Controlled growth chamber experiments were conducted to determine factors affecting seed germination and emergence of the troublesome weed Gomphrena perennis. The objective of this research was to examine the effects of temperature, light, moist chilling, osmotic potential, dry storage and depth of seed burial on G. perennis germination and emergence. The optimum temperature for germination was around 15–20°C. Seeds showed germination rates above 90% under 20/10 and 25/15°C temperature regimes. The minimum exposure to light needed to stimulate germination was 1 min. However, the light requirement was reduced after a long storage period. Furthermore, germination was high (>90%) in all moist‐chilling treatments tested. Germination was highly sensitive to increasing osmotic stress. The highest germination percentage (94%) was achieved at 0 MPa, and decreasing osmotic potential from 0 to ?0.3 MPa reduced germination to 11%. The highest seedling emergence occurred for seeds placed from 0 to 1 cm deep, and no seedlings emerged from a 5‐cm burial depth. Gomphrena perennis has a suitable environment in a no‐till soybean field, where seeds remaining on the surface have the required temperature, light and depth needed for germination.     

Purification and Characterization of 4-Hydroxyphenylpyruvate Dioxygenase from Maize

The proposed target enzyme for benzoylcyclohexanedione herbicides, 4-hydroxyphenylpyruvate dioxygenase (HPPD) was purified from etiolated maize seedlings with a purification factor of 105. Enzyme activity was measured by detection of carbon dioxide formed from radiolabelled substrate. The enzyme has a pH optimum of 7·3 and an apparent molecular mass of 43 kDa, similar to that of the mammalian liver enzyme. Activity needs the presence of a reducing system glutathione/dichlorophenol indophenol or ascorbate and catalase. Surprisingly, a commercial catalase preparation of low specific activity—generally used for the enzyme assay—showed HPPD activity which was separable from the catalase activity on a gel filtration column. According to kinetic studies with purified maize HPPD, experimental herbicides from the family mentioned were strong competitive inhibitors of the plant enzyme in nanomolar range withK_i values of 5 and 15 nM for 2-(2-nitro-4-chlorobenzoyl)-5-(2-methoxyethyl) cyclohexane- 1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)- cyclohexane-1,3-dione (SC-0051; sulcotrione), respectively.     

Enhanced protection against two major fungal pathogens of groundnut, <Emphasis Type="Italic">Cercospora arachidicola</Emphasis> and <Emphasis Type="Italic">Aspergillus flavus</Emphasis> in transgenic groundnut over-expressing a tobacco β 1–3 glucanase

Groundnut is an important oilseed crop of the Indian subcontinent. Yield losses due to fungal diseases are enormous in the cultivation of this crop. Over-expression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyses a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report in this paper, overexpression of a tobacco glucanase in transgenic groundnut and its resistance towards Cercospora arachidicola and Aspergillus flavus. PCR, Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in groundnut transgenics. When screened for resistance against Cercospora arachidicola the transgenics showed not only reduction in the number of spots but also delay in the onset of disease. Resistance was also demonstrated against one another important pathogen of groundnut, Aspergillus flavus. The transgenics not only resisted hyphal spread but also did not accumulate aflatoxin in the seeds. The results demonstrate the potential of a PR protein from a heterologous source in developing fungal disease resistant groundnut.     

Baseline and differential sensitivity of Peronophythora litchii (lychee downy blight) to three carboxylic acid amide fungicides

Downy blight, caused by Peronophythora litchii, is an important disease of lychee (litchi) plants in China. The in vitro sensitivities of various asexual stages of P. litchii to the three carboxylic acid amide (CAA) fungicides dimethomorph, flumorph and pyrimorph were studied with four single‐sporangium isolates. None of the three fungicides affected zoospore discharge from sporangia, but they strongly inhibited mycelial growth (mean EC₅₀ values of 0·075, 0·258 and 0·115 mg L^?1, respectively); sporangial production (mean EC₅₀ values of 0·085, 0·315 and 0·150 mg L^?1, respectively); germination of cystospores (mean EC₅₀ values of 0·140, 0·150 and 0·645 mg L^?1, respectively); and germination of sporangia (mean EC₅₀ values of 0·203, 0·5 and 0·743 mg L^?1, respectively). As mycelial growth was the most sensitive stage to dimethomorph and pyrimorph, it was chosen to test baseline sensitivities to the three fungicides. In 2007, from 131 isolates collected in Fujian, Guangdong and Guangxi provinces, 127, 116 and 113 isolates were used to establish baseline sensitivity for dimethomorph, flumorph and pyrimorph respectively. Isolates from different provinces exhibited similar baseline sensitivity to the same fungicide. Baseline sensitivities to dimethomorph, flumorph and pyrimorph were distributed as unimodal curves, with mean EC₅₀ values of 0·082 (± 0·01), 0·282 (± 0·047), and 0·115 (± 0·032) mg L^?1, respectively. This information will serve as a baseline for tracking future changes in sensitivities of P. litchii populations to these three CAA fungicides.