Inhibition of fungal spore adhesion by zosteric Acid as the basis for a novel, nontoxic crop protection technology

ABSTRACT To explore the potential for nontoxic crop protection technologies based on the inhibition of fungal spore adhesion, we have tested the effect of synthetic zosteric acid (p-(sulfo-oxy) cinnamic acid), a naturally occurring phenolic acid in eelgrass (Zostera marina L.) plants, on spore adhesion and infection in two pathosystems: rice blast caused by Magnaporthe grisea and bean anthracnose caused by Colletotrichum lindemuthianum. We have shown that zosteric acid inhibits spore adhesion to model and host leaf surfaces and that any attached spores fail to develop appressoria, and consequently do not infect leaf cells. Low concentrations of zosteric acid that are effective in inhibiting adhesion are not toxic to either fungus or to the host. The inhibition of spore adhesion in the rice blast pathogen is fully reversible. On plants, zosteric acid reduced (rice) or delayed (bean) lesion development. These results suggest that there is potential for novel and environmentally benign crop protection technologies based on manipulating adhesion.     

The spore coat of the bean anthracnose fungus Colletotrichum lindemuthianum is required for adhesion, appressorium development and pathogenicity

The spores (conidia) of the bean anthracnose fungal pathogen, Colletotrichum lindemuthianum, adhere to the aerial parts of plants to initiate the infection process. In previous studies we have shown that the Colletotrichum spores are surrounded by a fibrillar spore coat, comprising several major glycoproteins. Previous evidence showed that a monoclonal antibody (UB20) that recognised these glycoproteins was able to inhibit adhesion of spores to a hydrophobic surface. In this paper we have further studied the role of the spore coat in adhesion, germination and fungal development by studying the effects of UB20 and protease treatment of spores. The latter treatment has previously been shown to remove the spore coat. Spores germinate on glass, polystyrene and water agar, however, appressoria only develop on glass or polystyrene, showing a requirement for a hard surface. Removal of the spore coat with protease inhibits adhesion at 30 min, before the secretion of ECM glycoproteins. Protease treatment also inhibits the development of appressoria and reduces pathogenicity on leaves. Incubation of spores with the MAb UB20 inhibits adhesion at 30 min, but does not affect appressorium formation or pathogenicity. The results suggest that an intact spore coat has two functions; it is required for adhesion to a hydrophobic surface and for the detection of a hard surface necessary for appressorium formation. We suggest that contact with a hard surface, rather than adhesion, is the key event leading to appressorium formation.     

The effect of isoprothiolane on the emergence of infection pegs from appressoria of Pyricularia oryzae

The effect of isoprothiolane^a (di-isopropyl 1,3-dithiolan-2-ylidenemalonate), a systemic fungicide used for rice blast control, on the infection process of Pyriculuria oryzae was studied by micromanipulation in a scanning electron microscope. Whether or not infection pegs emerged from the appressoria was examined by inverting each appressorium contact surface. Infection pegs were observed on more than 80% of the appressoria, 48 h after inoculation, on both untreated rice leaves and cellophane film. Isoprothiolane, approximately 10 μg ml^?1 in the ambient water of inoculated conidia, reduced the emergence of infection pegs on rice leaves and cellophane film by 96 and 100%, respectively.     

Uptake and translocation of carpropamid in rice (Oryza sativa L)

Translocation of the antiblast compound, carpropamid, was investigated in rice using [¹⁴C]carpropamid. When applied to the seed, carpropamid was not only readily absorbed but was translocated to different parts of the seedlings emerging from treated seeds. A substantial portion of fungicide appeared to be exuded onto the leaf surface. In 21‐day‐old plants grown from [¹⁴C]carpropamid‐treated seeds, 27.2% of the radioactivity isolated from leaves was present on the surface of lamina. This exuded fraction is probably responsible for its action as a fungal anti‐penetrant compound. Following 30‐min root dipping of 14‐day‐old seedlings, carpropamid was rapidly absorbed and translocated throughout the seedling. Its intra‐laminar distribution was uniform as determined by autoradiography. Only a small fraction (<2%) of fungicide applied to the foliage was translocated beyond the site of application within the treated leaf. Translocation was primarily apoplastic. Approximately 54% of the radioactivity recovered from leaves was in the form of carpropamid. At least seven radiolabelled metabolic products were observed by TLC. Only 8.3% of radioactivity applied through the seeds could be recovered from 21‐day‐old seedlings. © 2001 Society of Chemical Industry     

Possible roles and functions of LPL1 gene encoding lysophospholipase during early infection by Magnaporthe grisea

The rice blast fungus Magnaporthe grisea differentiates appressoria, which are required to attack its rice plant host. Clone A26, tentatively named LPL1, was previously found to be homologous to the known lysophospholipase genes from our subtractive cDNA library. The LPL1 protein had a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence, and the protein expressed in Escherichia coli had lysophospholipase activity. To clarify the functions and possible roles of LPL1, the gene was disrupted by targeted gene replacement. The ΔLPL1 mutants formed fewer appressoria on the hydrophobic surface of GelBond film, and the appressoria had reduced turgor pressure and penetration into cells of the leaf sheath. The ΔLPL1 mutants and wild-type differentiated normal appressoria on other artificial substrata such as polycarbonate plate and on rice leaf sheath. Cytological analysis of the appressoria indicated that ΔLPL1 mutants had a delay in the disappearance of lipid droplets. These findings imply that LPL1, phospholipid metabolism, or both are involved in glycerol biosynthesis and accumulation to generate turgor pressure in the appressorium. LPL1 was, however, dispensable for full pathogenicity, suggesting that other complementary pathways or similar genes related to phospholipid metabolism probably function in M. grisea.     

Patterns of Splash Dispersed Conidia of Fusarium poae and Fusarium culmorum

Splash dispersal of Fusarium culmorum and Fusarium poae spores was studied, using inoculated straw placed on tiles as the inoculum source to infect agar strips and artificially produced leaves. In addition, patterns of spread were studied with spores from inoculated artificial leaves onto agar strips. Observed patterns of spore dispersal for each species were indistinguishable, although F. culmorum produced fewer colonies than F. poae. Furthermore, spore dispersal from inoculated straw and artificial leaves were essentially identical, with one exception; colonies arose from single conidia when spread from artificial leaves, but consisted of clumps of conidia when derived from inoculated straw. Splash dispersal patterns of both species onto the upper- and undersides of artificial leaves were different. On the upperside of the leaf, most colonies were found at the tip, while on the underside of the leaf most colonies were found at the base of the leaf. This is the first time that artificially produced leaves have been used in splash dispersal experiments.     

三环唑对稻瘟病菌再侵染的抑制作用初探

在实验室条件下模拟稻瘟病菌Magnaporthe grisea再侵染发生的全过程,研究了三环唑对稻瘟病菌再侵染的抑制作用及其机制。在接种稻瘟病菌48 h后喷雾处理,10 μg/mL三环唑的防病效果只有28.58%,表明三环唑对稻瘟病的治疗效果较差;10 μg/mL三环唑处理过的发病植株作为再侵染源引起周围未经药剂处理的健康水稻的发病程度要比未经药剂处理的病株作为再侵染源引起的低63.28%。进一步的研究表明,三环唑抑制稻瘟病菌再侵染的主要机制是抑制再侵染源的孢子产生和降低分生孢子的释放及致病能力。     

The role of silicon in preventing appressorial penetration by the rice blast fungus

To test the hypothesis that silicon (Si) confers resistance against appressorial penetration of the rice blast fungus, the proportion of appressorial penetration into the leaf epidermis to total appressoria formed was compared among rice plants amended with various rates of silica gel to those plants nonamended. The amounts of Si in the youngest leaves were consistent with the amounts of silica gel applied to the rice plants. Relative Si levels on the adaxial surface of leaves as detected by energy dispersive X-ray analysis also increased with the amounts of silica gel applied. Based on light microscopic observation of the adaxial surface of rice leaves, the proportion of appressorial penetration was reduced by increasing amounts of silica gel applied and increased with the length of period after spray inoculation. Consequently, these results strongly support the hypothesis and suggest that Si in the leaf epidermis may confer resistance against appressorial penetration. Meanwhile, the number of lesions per leaf also decreased with the amount of Si applied, while only a certain part of penetrated appressoria could become sporulating susceptible lesions. This suggests that Si also confers physiological resistance against blast infection after the penetration.     

稻梨孢菌与其他寄主梨孢菌在水稻植株上的交互作用

作者研究了1个稻梨孢菌株与4个稻以外寄主梨孢菌株在混合接种和间隔接种条件下,在水稻植株上的交互作用。结果表明:弱致病菌与强致病菌间交互作用较强,非致病菌与强致病菌间较弱。弱致病菌可明显降低强致病菌的致病性,非致病菌对强致病菌的作用,多数组合表现为减轻病害,但有少数组合表现为促进发病。弱致病菌先接种;2天后接种强致病菌,比两者混合接种具有更强烈的互作效应,病斑数减少达35.7%-38.1%。先接种菌的孢子浓度对后接种菌的致病性也有影响。     

Comparative colonisation by virulent versus avirulent <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> on wild <Emphasis Type="Italic">Oryza australiensis</Emphasis>

Pyricularia oryzae (rice blast) conidial development at pre-penetration stage determines success or otherwise of infection inside the rice host plants. Studies on conidial germination and growth on the leaf surface in commercial rice (Oryza sativa) report differently, dependent upon host type and level of blast resistance. Although wild rice (O. australiensis) is known to be an alternative host of blast, the interaction between P. oryzae conidia and wild O. australiensis on its leaf surface has not been previously studied. We found significant (P?<?0.001) differences in conidial development between two blast isolates with different virulence in terms of conidial germination, germ tube growth and appressoria formation on both wild and cultivated rice. Conidial germination at 6 h post-inoculation (hpi) for the virulent isolate was significantly (P?<?0.001) delayed. Germ tubes of the avirulent isolate conidia grew significantly (P?<?0.001) faster and with significantly (P?<?0.001) longer germ tubes than from virulent conidia. Appressoria development for the virulent isolate was significantly (P?<?0.001) faster at its later growth stages of 12 and 18 hpi when approximately 100% of germ tubes formed appressoria. In contrast, formation rate of appressoria for the avirulent isolate was significantly (P?<?0.001) slower and only reached 76% of germ tubes forming appressoria. Appressoria formation on O. australiensis was significantly (P?<?0.001) greater than the formation on O. sativa for both virulent and avirulent P. oryzae at 12 hpi, a clear indication that host type influences the extent of appressoria formation.     

烟草黑胫病菌培养特性的研究

研究了云南烟草黑胫病菌在不同培养基、不同温度的生长情况及不同菌龄与不同诱导时间孢子囊的产生数量。结果表明,菌株在8种供试培养基中,以燕麦培养基和选择性培养基上菌落生长最好,其次为胡萝卜培养基,在番茄汁和马铃薯培养基上生长最差。同一菌株在不同培养基上产孢量不同、产生的孢子囊大小亦有所不同。05P226菌株在玉米培养基上产孢量最高;05P55菌株在燕麦培养基上产孢量最高,在番茄汁培养基上产生的孢子囊最大,而05P62菌株在燕麦培养基上产孢量最高,在烟叶汁培养基上产生的孢子囊最大。在30℃菌丝生长速率最高,其次为28℃,以35℃生长最差。供试3个菌株间生长差异显著,以05P226生长最好,05P55菌株次之,05P62生长最差。05P226、05P55菌株皆以培养14d、诱导72h产孢量最高,最适宜试验中产孢培养。     

Transmission of rice blast from seeds to adult plants in a non‐systemic way

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a serious threat to rice production worldwide. In temperate regions, where rice is not cultivated for several months each year, little is known about the initial onset of the disease in the field. The main overwintering and primary inoculum sources reported are infested residues and seeds, but the subsequent steps of the disease cycle are largely unknown, even though a systemic infection has been proposed but not demonstrated. The present work follows rice blast progression in infected seeds from germination to seedling stage, with direct and detailed microscopic observations under both aerobic conditions and water seeding. With the use of GFP‐marked M. oryzae strains, it was shown that spores are produced from contaminated seeds, infect emerging seedling tissues (coleoptile and primary root) and produce mycelium that colonizes the newly formed primary leaf and secondary roots. Using different rice cultivars exhibiting distinct levels of resistance/susceptibility to M. oryzae at the 2/4‐leaf stage, it was observed that resistance or susceptibility of a considered genotype is already established at the seedling stage. The results also showed that when plants are inoculated either at ripening stage (mature panicles), heading stage (flowering/immature panicles) or even before heading (flag leaf fully developed), they produce infested seeds. These seeds produce contaminated seedlings that mostly die and serve as an inoculum source for healthy neighbouring plants, which gradually develop disease symptoms on leaves. The possible rice blast disease cycle was reconstructed on irrigated rice in temperate regions.     

Effects of leaf blast on photosynthesis of rice. 1. Leaf photosynthesis

The effect ofPyricularia oryzae, the causal organism of leaf blast in rice, on photosynthesis characteristics of rice leaves was measured in two greenhouse experiments. Leaf blast reduced photosynthesis not only through a reduction in green leaf area, but also through an effect on photosynthesis of green leaf tissue surrounding the lesions. The assimilation rate at light saturation (P _max ) was more affected than the initial light use efficiency (). Dark respiration (R _d ) increased as a result of infection. The experimental data were used to derive relations between leaf blast severity andP _max , andR _d .     

枯草芽孢杆菌B-332菌株对稻瘟病的防治效果及定殖作用

为了进一步明确枯草芽孢杆菌B-332菌株对稻瘟病的生防作用,分别对盆栽水稻的防治效果、防治适期、标记菌株在水稻上的定殖作用及田间的防治效果进行了考察。结果表明,在盆栽水稻活体生物测定中,B-332的芽孢菌体悬浮液及发酵上清液对稻瘟病均表现出较好的防治效果(50%以上),且当菌液与上清液混合后其防治效果有加和作用。该菌株对稻瘟病的防治作用主要体现在预防,其预防叶瘟的效果与接种稻瘟病菌时叶面已定殖的B-332菌株的数量有关,接种时水稻叶面上定殖的菌量为1.25×104cfu/mm2时,其防治效果可以达到45%。在人工大量引入的情况下,该菌能够在水稻叶面上有效定殖达21天以上,并且在叶面有10%病斑的情况下,检测到的标记菌株数量会更多。田间试验结果表明,芽孢浓度在1×108cfu/mL时,该菌株对水稻穗颈瘟的防治效果达57.2%,增产率达9.6%,且浓度在5×108cfu/mL时对螃蟹无毒性。     

Vertical distribution of leaf blast lesions in mixtures of rice cultivar Sasanishiki and its resistant near-isogenic line

The vertical distribution of leaf blast lesions caused by the fungus Pyricularia grisea was studied to estimate the degree of leaf blast suppression in rice multilines in experimental paddy fields for 4 years. Leaf blast in 1 : 1 and 1 : 3 mixtures of susceptible rice cultivar Sasanishiki and its resistant near-isogenic line, Sasanishiki BL7, developed slower than that in pure stands of Sasanishiki. The average distance of lesions on leaves from the ground in the 1 : 3 mixtures was significantly lower than that in the pure stands at the end of leaf blast epidemics (at booting stage). This result shows that the distribution of leaf blast lesions in the upper layer differs between the susceptible pure stands and the 1 : 3 mixtures at the end of leaf blast epidemics.     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

Resistance in leaf blades assessed by counting conidia correlates with whole-plant-specific resistance in leaf sheaths in a compatible rice–Magnaporthe oryzae interaction

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.     

Life-table analysis of faba bean rust

In controlled near-optimum conditions (18 °C), monocyclic sporulation capacity and spore infection efficiency were assessed for faba bean rust on the first and second leaves of field bean. After a latency period of 8–10 days, lesions sporulated duringc. 50 days. Spore production on the second leaf,c. 9×10⁴ spores per lesion, was two times as high as spore production on the first leaf. Infection efficiency was similar for both leaf layers, with a mean value of 0.11 lesion per inoculated spore. Infection efficiency decreased strongly when spores originated from mother lesions older than 20 days. Three life-table statistics (the net reproduction numberR _o , the mean generation timeT _g , and the maximum relative growth rater _max ) were calculated.R _o was larger andT _g was longer for the second than for the first leaf, butr _max was nearly the same for both leaf layers (0.31–0.33 day^–1).r _max was compared with the exponential growth rater measured in a field experiment. From the difference between the two rates, the fraction of inoculum lost in field conditions was estimated at 0.54–0.94. The life-table statistics were also compared to those of other legume rusts, and implications of life-table analysis for comparative epidemiology were discussed.     

Pathogenicity,host range and activities of a secondary metabolite and enzyme from Myrothecium roridum on water hyacinth from Thailand

Leaf blight disease of water hyacinth was observed and collected from different geographical areas of Thailand. The disease is caused by a fungal pathogen that was identified as Myrothecium roridum by using its morphological characteristics. The most effective fungal strains were evaluated for pathogenicity on water hyacinth under greenhouse and natural conditions. Myrothecium roridum isolate, Kamphaeng Sean Campus (KKFC) 448, was found to be the most virulent. Different fungal formulations were evaluated for their level of control of water hyacinth. The results showed that spore suspensions with 10% palm oil or 1% Tween 20 caused a higher level of disease severity, compared to spores applied in water alone. The host range of KKFC 448 was evaluated by using 77 plant species that belong to 40 plant families. The fungus did not cause disease on 74 economically important plants but did produce disease signs on water hyacinth and two other aquatic weeds, duckweed and water lettuce. Leaf blight occurs on water hyacinth leaves after being treated with crude extracts of M. roridum and it was indicated that secondary metabolites were released from the fungal mycelia. Myrothecium roridum that was grown on boiled paddy rice produced β‐1,4‐exoglucanase, β‐1,4‐endoglucanase, β‐glucosidase, xylanase and pectinase more than Mroridum that was grown on potato dextrose agar. The results indicated that M. roridum is a pathogen of water hyacinth and the fungus is capable of producing different enzymatic activities on potato dextrose agar and boiled paddy rice, which might be important for infection.     

Specific inhibition of spore germination of <Emphasis Type="Italic">Alternaria brassicicola</Emphasis> by fistupyrone from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. TP-A0569

Fistupyrone (FP), a metabolite from Streptomyces sp. TP-A0569, inhibited the in vivo infection of Chinese cabbage seedlings by Alternaria brassicicola. To detect the possible action sites of FP, the effect of FP on the infection behavior of A. brassicicola and A. alternata was investigated. When spores of A. brassicicola were suspended in FP solution and inoculated on host leaves, FP at 0.1ppm significantly inhibited spore germination, appressorial formation, and infection hypha formation of A. brassicicola. Host-specific AB-toxin production and lesion formation by A. brassicicola spores were also reduced significantly by treatment with FP 1ppm. The effect of FP seemed to be irreversible because significant washing of FP-treated spores with distilled water (DW) did not change the inhibitory effects. In contrast, A. alternata isolates such as Japanese pear pathotype, apple pathotype, and saprophyte behaved almost equally in both FP- and DW-treated spores. Mycelial dry weight in potato dextrose broth and mycelial diameters on potato dextrose agar, gelatin glucose agar, and Czapek solution agar of both A. brassicicola and A. alternata were not different with or without addition of FP. These results indicate that FP at low concentrations has a fungicidal effect on spores of A. brassicicola but not on spores of A. alternata; FP also does not affect the vegetative phase of these fungi.