Inhibition of mycelial growth and sporidial formation in the wheat pathogenNeovossia indica by a polyamine biosynthetic inhibitor

DL-α-Difluoromethylornithine (DFMO), a specific suicide inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly inhibited mycelial growth and sporidial formation of the wheat pathogen,Neovossia indica, in vitro, while DL-α-difluoromethylarginine (DFMA), the analogous suicide inhibitor of arginine decarboxylase (EC 4.1.1.19), did not. The inhibited mycelial growth and sporidial formation were not only restored by putrescine (polyamine) addition, but were actually enhanced in the putrescine + DFMO cultures. Besides altering mycelial growth and morphology, DFMO also reduced the cell size drastically. The inhibition of fungal polyamine biosynthesis is discussed in relation to selective control of plant disease.     

Calcium/calmodulin-dependent signaling for prepenetration development in <Emphasis Type="Italic">Cochliobolus miyabeanus</Emphasis> infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca²⁺ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.     

The development of rhizomania in two areas of the Netherlands and its effect on sugar-beet growth and quality

Since detection of beet necrotic yellow vein virus (BNYVV) by serological techniques was not possible 10 years ago, there is no relaible information about the occurrence of the disease in the Netherlands prior to 1983. By evaluating historical information on the sugar production of fields, which have been proved to be infested by rhizomania recently, the time of occurrence of the first infections could be estimated roughly.Analysis of root samples from diseased fields and comparison with uninfected fields, revealed a correlation between sugar and sodium content and the occurrence of rhizomania. Therefore both quality parameters can be used as indicators for the disease under Dutch conditions.Differences between infected and uninfected fields were detected from mid-June onwards. Only a weak correlation between sugar and -amino N content could be established. At later stages of crop growth the -amino N level of diseased fields was always lower than that of healthy ones. However sugar content remained at a low level and did not increase substantially.Samenvatting Aangezien tien jaar geleden geen serologische technieken beschikbaar waren, kon het voorkomen van bieterhizomanie in Nederland niet worden aangetoond. Door het beoordelen van de suikeropbrengsten van percelen die met bieterhizomanie besmet bleken te zijn, over een reeks voorafgaande jaren, kon een periode waarin de infectie vermoedelijk had plaatsgevonden worden vastgesteld.Door analyse en vergelijking van bietmonsters van een reeks aangetaste proefplekken te een aantal gezonde referentiepercelen, kon worden aangetoond dat onder Nederlandse omstandigheden suiker- en Na-gehalten de meest betrouwbare indicatoren voor de aanwezigheid van bieterhizomanie zijn.Afwijkingen in de gehalten van deze kwaliteitsparameters werden in de zwaar aangetaste percelen vanaf half juni gevonden. Er werd een zwakke relatie tussen de gehalten aan suiker en -amino N aangetoond. Gedurende de tweede helft van de zomer was het gehalte aan -amino N in de zieke percelen lager dan dat van bieten in de gezonde. Het suikergehalte bleef op een laag niveau en vertoonde geen of een geringe toename.     

An inhibitor of polyamine biosynthesis — Difluoromethylornithine — and the polyamine spermidine for the control of gray mold(Botrytis Cinerea)

Difluoromethylornithine (DFMO) — an inhibitor of polyamine biosynthesis, and the polyamine spermidine (Spd) reduced gray mold of tomato, pepper, eggplant, bean andSenecio sp. leaves, and of rose petals by 37–88% when applied at 0.1–1.0 mM each. Higher doses did not result in better control. The disease was also reduced significantly on tomato fruits by 1.0 inM DFMO and by 0.1–1.0 mM Spd, and on cucumber fruits by 0.1–1.0 mM of both compounds, but not on grape berries. The combination of 0.2 mM DFMO with 1.0 mM Spd controlled gray mold ofSenecio sp. and tomato leaves additively better than either treatment alone, whereas this effect was not observed in leaves of lettuce and pepper. Ethylene production was reduced significantly by Spd applied to leaves of tomato and pepper, but not by DFMO. Linear growth and germination of the fungus were affected by lower concentrations of DFMO (ED50 0.12–0.97 and 1.4, respectively) as compared with Spd. Spermidine and DFMO controlled white mold(Sclerotinia sclerotiorum) as effectively as did the fungicide benomyl. Contribution from the Agricultural Research Organization. No. 3195-E, 1991 series.     

Biological Mode of Action of the Fungicide, Flusulfamide, Against Plasmodiophora brassicae (clubroot)

Flusulfamide (2, 4-dichloro-,,-trifluoro-4-nitro-m-toluenesulfonanilide) was investigated for its mode of action against Plasmodiophora brassicae Woronin. Seedlings of Chinese cabbage (Brassica rapa L. subsp. pekinensis) were grown for 14 and 21 days in soil infested with P. brassicae and then transplanted into soil containing flusulfamide (0.9µg a.i.g^–1 dry soil). Clubroot was not suppressed by this treatment, indicating that the fungicide is ineffective against P. brassicae established within cortical cells of the host root. Where seedlings were grown in soil infested with resting spores which had previously been treated with flusulfamide, root-hair infection and club formation were suppressed. This indicates that flusulfamide directly acts against resting spores. When placed in root exudates of Chinese cabbage, untreated resting spores germinated at a high frequency while flusulfamide-treated resting spores hardly germinated at all. Use of the Evan's blue staining assay indicated that flusulfamide-treated resting spores remained viable. Flusulfamide was detected by high performance liquid chromatography on resting spores treated with flusulfamide for 30min. This indicates that the chemical is adsorbed onto resting spores. These results suggest that flusulfamide suppresses clubroot disease by inhibiting germination of P. brassicae resting spores through adsorption onto their cell walls.     

Effects of α-difluoromethylornithine on photosynthetic oxygen evolution,respiration and polyamine concentrations in detached barley leaves

Detached barley leaves were treated with the polyamine biosynthesis inhibitor, α-difluoromethylornithine (DFMO; 1 mM ) for either 30 min or 12 h. Exposure to DFMO for 30 min led to a substantial reduction in photosynthesis, expressed either on a unit leaf-area or unit chlorophyll basis. On the other hand, photosynthesis (expressed on a unit chlorophyll basis) was increased in leaves exposed to DFMO for 12 h, although when expressed on a unit leaf-area basis, photosynthesis remained unchanged at lower photon flux densities (0-300 μmol m^?2) and was slightly reduced at higher photon flux densities (300-800 μmol m^?2 s^?1) s^?1). Respiration was unaltered in leaves exposed to DFMO for 30 min or 12 h. Exposure to DFMO for 30 min resulted in significant increases in chlorophyll concentrations, while carotenoid concentrations remained unaltered. However, chlorophyll concentrations were reduced in leaves treated with DFMO for 12 h, although no change in carotenoid concentration was observed. Polyamine concentrations were not significantly affected by exposure of detached barley leaves to DFMO for 24 h.     

Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.     

Abscisic acid is a potent inhibitor of growth and sporidial formation inneovossia indica cultures: Dual mode of actionvia loss of polyamines and cellular turgidity

The inhibition of polyamine biosynthesis inNeovossia indica (Mitra) Mundkur by D,L— α-difluoromethylornithine (DFMO) caused an effective reduction of mycelial growth and sporidial production underin vitro conditions, which was reversed by ornithine application. Abscisic acid proved to be similarly effective, not only in inhibiting mycelial growth and sporidial formation but also the germination of teliospores, which constitute the primary inoculum of the pathogen. ABA-mediated inhibition resulted in decreased polyamine levels and loss of cellular turgidity of mycelial cultures. Scanning electron microscopy of ABA-treated cultures revealed extremely shrunken hyphae, in marked contrast to the turgid controls. It is suggested that the manipulation of ABA levels and/or tissue sensitivity in wheat could be a strategy to combat ‘Karnal’ bunt, the disease caused byN. indica.     

疏水表面诱导橡胶树炭疽菌侵染结构的发育分化过程

为了解橡胶树2种炭疽病菌的侵染结构发育分化过程,采用平板菌落生长速率法测定了3株胶孢炭疽菌Colletotrichum gloeosporioides和3株尖孢炭疽菌C.acutatum的菌丝生长速率,测量其分生孢子大小,显微观察2种炭疽菌在疏水表面诱导下侵染结构的发育分化过程。结果表明,胶孢炭疽菌菌丝生长速率为0.96~1.36 cm/d,显著高于尖孢炭疽菌的菌丝生长速率0.72~0.89 cm/d,但二者分生孢子大小无显著差异。在疏水表面诱导下,2种炭疽菌分生孢子在接种2~6 h后开始萌发,12 h孢子萌发率为71.70%~88.05%,13~16 h开始分化附着胞,24 h附着胞形成率为48.99%~70.74%,36 h菌丝诱发形成大量附着枝,48 h后分生孢子产生的次生菌丝也可诱发形成附着枝,附着枝呈圆形、姜瓣形、梨形或不规则形。分生孢子极易产生,可在菌丝顶端成簇或菌丝侧面排列产生,也可由分生孢子形成的芽管产生,或在芽管分化附着胞过程分枝形成分生孢子;附着胞多着生于芽管顶端,少数附着胞顶端可继续萌发类似短芽管结构,再次分化形成可黑色化的次级附着胞。表明橡胶树2种炭疽菌不同菌株间分生孢子萌发时间、孢子萌发率、附着胞形成时间和形成率有一定差异,但种间无明显差异;橡胶树炭疽菌分生孢子极易形成,在疏水表面容易分化形成附着胞和附着枝,说明具有极强的适生性。     

Characterisation of the saponin hydrolysing enzyme avenacoside-α-l-rhamnosidase from the fungal pathogen of cereals, Stagonospora avenae

The fungal pathogen Stagonospora avenae f. sp. avenaria infects oat leaves, which contain the saponins avenacoside A and B. The avenacosides are glycosylated steroidal saponins that occur within oat leaves in a non-fungitoxic form and are converted upon damage or pathogen invasion to their antifungal form by a plant enzyme. It has previously been shown that oat-attacking isolates of S. avenae are able to hydrolyse the sugar chain at the C3 position of the avenacosides. This carbohydrate moiety is a branched chain that consists of one -{scL}-rhamnose and two or three -{scD}-glucose residues in avenacosides A and B, respectively. Removal of the -{scL}-rhamnose residue is sufficient to detoxify the avenacosides. This work describes the purification of the avenacoside-degrading -{scL}-rhamnosidase–and determination of peptide sequence from the protein which represents the first -{scL}-rhamnosidase thus characterised in a fungal plant pathogen.     

Inhibition of polyamine biosynthesis in Phytophthora infestans and Pythium ultimum

Mycelial growth, polyamine concentrations and the activities of enzymes of polyamine biosynthesis and catabolism were examined in Phytophthora infestans and Pythium ultimum grown in the presence of difluoromethylornithine (DFMO), an inhibitor of omithine decarboxylase (ODC), and difluoro-methylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC). Growth of P. infestans was reduced by DFMO and a mixture of DFMO + DFMA, but was increased by DFMA at concentratiotis of 5 MM and greater, Polyamine concentrations and ODC activity were significantly reduced in P. infestans grown on all inhibitor treatments. In contrast, growth, polyamine concentrations and enzyme activities were not affected in P, ultimum exposed to the inhibitors.
It seems unlikely that P. infestans and P. uttimum possess ADC activity, as neither of the products of ADC activity (agmatine and putrescine) could be detected in ADC assays. Although ODC from P. infestans was sensitive to DFMO, ODC from P. uttimum was insensitive to the inhibitor. Moreover, uptake of DFMO by P. infestans was three times greater than that observed by P. ultimum.     

Elicitor(s) production is involved in red-light-induced resistance during spore germination of <Emphasis Type="Italic">Bipolaris oryzae</Emphasis> in the presence of host tissues

In the present study, the effect of red light on the infection behavior of Bipolaris oryzae on rice leaves and the effects of chemical inhibitors and spore germination fluid (SGF) of B. oryzae on red-light-induced resistance against brown spot disease were investigated. Red light irradiation and natural light did not differ significantly with respect to their effect on spore germination and appressorium formation of B. oryzae 24 h after inoculation. However, formation of infection hyphae was significantly inhibited under red light compared to that under natural light. Pretreatment with the photosynthetic inhibitor 3-(3,4-dichorophenyl)-1,1-dimethylurea (DCMU) or with the aromatic amino acid inhibitor N-(phosphonomethyl) glycine (glyphosate) for 24 h inhibited the development of resistance in rice leaves. To elucidate the elicitor(s) produced during the B. oryzae–rice plant interaction, the effect of SGF prepared in the absence (A-SGF) or presence (P-SGF) of rice leaves on red-light-induced resistance was investigated. When rice plants were pretreated with A-SGF or P-SGF for 24 h, P-SGF had elicitor activity under red light, but not under natural light or with A-SGF. These results suggest that germinating spore of B. oryzae produced an elicitor(s) under red light conditions in a host-dependent manner. In conclusion, we hypothesize that an unidentified elicitor(s) produced at an early stage of fungal infection during the B. oryzae–rice interaction contributes to the inhibition of cell death in rice leaves under red light and enhances resistance-related tryptophan and phenylpropanoid pathways.     

ZJUF0986活性代谢产物对稻瘟病菌致病性的影响

通过菌丝生长速率法和悬滴法测定ZJUF0986活性代谢产物对稻瘟病菌菌丝生长、孢子萌发和附着胞形成的影响。结果表明,ZJUF0986活性代谢产物对稻瘟病菌菌丝生长的有效中浓度EC50为18.55mg/L,与对照药剂三环唑的EC50(17.30mg/L)相比无显著性差异。活性代谢产物不仅影响孢子萌发,也显著降低附着胞的形成。浓度为10mg/L的活性代谢物可完全抑制孢子萌发及附着胞的形成;浓度为1.25mg/L时则明显延缓孢子萌发及附着胞的形成,处理48h后的孢子萌发率和附着胞形成率分别为62.17%和38.46%。以浓度为1.25mg/L活性代谢产物处理的稻瘟病菌孢子悬浮液接种离体大麦叶片,病原菌孢子在大麦叶片表面能部分萌发形成附着胞,但侵染栓形成延迟,致病性明显降低。     

Ulvan effect on conidial germination and appressoria formation of Colletotrichum gloeosporioides

Ulvan is an algal polysaccharide known for its ability to induce resistance to plant diseases such as the Glomerella leaf spot of apple caused by Colletotrichum gloeosporiodes. This study was aimed at investigating microscopically, in tests in vitro and in vivo, whether ulvan interferes in the development of pre-infective structures of C. gloeosporioides. Conidial germination and appressoria formation were monitored hourly on agar and cellophane, and at 48 h on water- and ulvan-treated susceptible as well as resistant apple leaves. Amendment of agar with ulvan (10 mg ml^?1) enhanced the germination and resulted in longer germ tubes at 7 h of incubation. On cellophane it significantly delayed appressoria formation up to 8 h, but later after 14 h increased the number of appressoria per conidium. Spraying of susceptible leaves with ulvan 6 days before inoculation decreased disease severity by 50%. This was associated with inhibition of appressoria formation and stimulus in growth of germ tubes, without interfering with conidial germination, when compared with both water-treated control and resistant plants. Appressorium formation occurred preferentially on anticlinal walls of epidermal cells and its location was not influenced by host resistance or by ulvan treatment. This study suggests a new mode of action for ulvan interfering with appressorium formation that could protect apple plants against C. gloeosporioides infection.     

Acibenzolar- S -methyl-induced resistance to sunflower rust (Puccinia helianthi) is associated with an enhancement of coumarins on foliar surface

Exogenous applications of acibenzolar- S -methyl (ASM) induced resistance to rust infection in sunflower, characterized by reduced infection frequency with no effect on latency period or pustule size, and no increase in host cell necrosis. Cytological studies showed that the reduced frequency of infection was due to a reduction in germination and appressorium formation, while stoma penetration, growth of infection hyphae and haustorium formation remained unaffected. Germination and germtube growth were not hampered by the direct application of ASM on urediospores. The data suggested that ASM had an effect on the production and secretion of fungitoxic compounds to the leaf surface that hamper rust urediospore germination and appressorium formation. This hypothesis is supported by the following experimental results: (i) an increase in the amount of accumulated and excreted coumarins and other phenolic compounds in ASM-treated plants, and (ii) a reduction of germination and of appressorium formation when ayapin, scopoletin, and leaf exudates collected from ASM-treated plants were applied exogenously.     

Calcium/Calmodulin-Dependent Signaling for Prepenetration Development in Colletotrichum gloeosporioides

ABSTRACT Colletotrichum gloeosporioides forms a specialized infection structure, an appressorium, for host infection. Contacting hard surface induces appressorium formation in C. gloeosporioides, whereas hydrophobicity of the contact surface does not affect this infection-related differentiation. To determine if the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper, effects of calcium chelator (EGTA), phospholipase C inhibitor (neomycin), intracellular calcium modulators (TMB-8 and methoxy verampamil), and calmodulin antagonists (chloroproma-zine, phenoxy benzamine, and W-7) were tested on conidial germination and appressorium formation. Exogenous addition of Ca(2+), regardless of concentration, augmented conidial germination, while appressorial differentiation decreased at higher concentrations. Inhibition of appressorium formation by EGTA was partly restored by the addition of calcium ionophore A23187 or CaCl(2). Calcium channel blockers and calmodulin antagonists specifically reduced appressorium formation at micromolar levels. These results suggest that biochemical processes controlled by the calcium/calmodulin signaling system are involved in the induction of prepenetration morphogenesis in C. gloeosporioides pathogenic on red pepper.     

Metabolism of phaseollin by different races of Colletotrichum lindemuthianum

The ability of four races of the bean pathogenColletotrichum lindemuthianum to metabolize the phytoalexin phaseollin in shake cultures was compared. Apart from some differences in the rate of conversion, all races metabolized the phytoalexin in the same way. Phaseollin was first converted to 6a-hydroxyphaseollin, and this product was further metabolized to 6a, 7-dihydroxyphaseollin. No metabolites of the latter compound could be detected.6a, 7-Dihydroxyphaseollin was as inhibitory as phaseollin to race 11, but was only slightly inhibitory to races 1, 2 and 1.Samenvatting Een vergelijkend onderzoek werd verricht naar het vermogen van vier fysiologische rassen van het bonepathogeenColletotrichum lindemuthianum om het fytoalexine phaseolline om te zetten.In schudculturen waaraan 10 g phaseolline/ml was toegevoegd, werd dit door alle fysio's op gelijke wijze omgezet, hoewel met verschillende snelheid (Fig. 1). Steeds werd phaseolline eerst omgezet tot 6a-hydroxyphaseolline, en dit produkt vervolgens tot een verbinding die geïdentificeerd kon worden als 6a, 7-dihydroxyphaseolline. Hierna konden geen verdere produkten worden aangetoond.6a, 7-Dihydroxyphaseolline was even fungitoxisch als phaseolline voor fysio 11, maar was slechts weinig fungitoxisch voor de fysio's 1, 2 en 1 (Tabel 1).De verschillen in omzettingssnelheid van phaseolline en in gevoeligheid voor phaseolline ee zijn omzettingsprodukten die tussen de fysio's gevonden zijn, zijn onvoldoende om de fysiospecifieke interacties tussen de boon en de verschillende fysio's vanC. lindemuthianum te verklaren.     

Eugenol reduces growth and increases activity of S-adenosylmethionine decarboxylase in the phytopathogenic fungusBotrytis fabae

This study was undertaken to examine the possibility that eugenol-induced reductions in growth ofBotrytis fabae are associated with alterations in polyamine metabolism.B. fabae was grown in liquid medium amended with different concentrations of eugenol. Changes in fungal biomass, and activities of enzymes of polyamine biosynthesis and catabolism were studied. An examination was also made of the incorporation of radioactivity from ornithine into polyamines. Activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC) and flux of label from ornithine into the polyamine spermine were greatly increased inB. fabae grown in the presence of eugenol. However, no significant changes were observed in polyamine catabolism or in the concentrations of free polyamines in treated fungal tissue. http://www.phytoparasitica.org posting May 17, 2005.