Appresoorium formation of Puccinia hordei on partially resistant barley and two non-host species

One of the components of partial resistance of barley to leaf rust,Puccinia hordei, is a reduced infectibility. It was investigated whether this low infectibility may rest on a hampered appressorium formation of the leaf rust fungus. The appressorium formation on the primary leaves of 11 barley genotypes with an intermediate-to-low infectibility was compared with that on the highly infectible L94. The number of stomata per cm² leaf area occupied by appressoria ofP. hordei was determined per genotype by means of fluorescence microscopy. No cosistent differences could be detected, indicating that the mechanisms causing a low infectibility of partially resistant barley seedlings act at a phase later than the formation of the appressoria. On the non-host wheat not fewer appressoria were formed than on L94, but no appressoria were found on a lettuce genotype. The latter probably lacks the stimuli that enable the fungus to find stomata.Samenvatting Eén van de componenten van partiële resistentie van gerst tegen dwergroest,Puccinia hordei, is een verminderde infectiedichtheid. Het mechanisme, dat hieraan ten grondslag ligt, is onbekend. Een experiment werd uitgevoerd om na te gaan of bij partieel resistente rassen een verminderde appressoriumvorming optreedt. Na inoculatie in een inoculatietoren en een zorgvuldig uitgevoerde incubatie werd het aantal huidmondjes per cm² bladoppervlak bepaald dat bezet was door appressoria vanP. hordei. De elf weinig vatbare gerstlijnen uit deze studie bleken niet reproduceerbaar te verschillen van de zeer vatbare gerstlijn L94 in de mate van appressoriumbezetting. Dit wijst erop dat infectiedichtheidsverschillen t.g.v. partiële resistentie veroorzaakt worden door mechanismen die werken na de appressoriumvorming. In een tweede experiment werd aangetoond dat zelfs op de niet-waardsoort tarwe, waaropP. hordei geen symptomen veroorzaakt, niet minder appressoria worden gevormd dan op L94. Op een sla-genotype trad echter geen appressoriumvorming op. Op deze laatste niet-waardsoort ontbreken waarschijnlijk de stimuli die de schimmel in staat stellen huidmondjes te vinden.     

Germination and appressorium formation of wheat leaf rust on susceptible,partially resistant and resistant wheat seedlings and on seedlings of other Gramineae

Germination and appressorium formation of wheat leaf rust urediospores were studied in two experiments. No consistent differences could be detected between 11 genotypes of wheat (Triticum aestivum), two of barley, one ofTriticum dicoccum, one ofT. dicoccoides, one ofT. boeoticum and one ofAegilops squarrosa. Host genotypes and non-hosts gave similar results, suggesting that the phases before penetration of the host leaf are not affected by the resistance mechanisms operating in hosts and non-host genotypes.Samenvatting Kieming en de vorming van appressoria is bestudeerd in twee experimenten. Er zijn geen systematische verschillen waargenomen tussen 11 genotypen van tarwe, twee van gerst, een vanTriticum dicoccum, een vanT. dicoccoides, een vanT. boeoticum en een vanAegilops squarrosa. Waard en niet-waard genotypen gaven overeenkomstige resultaten, dit suggereert dat de fasen voor penetratie van het gastheerblad niet beïnvloed worden door de resistentiemechanismen werkzaam in waard en niet-waard.     

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。     

Volatile organic compounds (VOCs) from cereal plants infested with crown rot: their identity and their capacity for inducing production of VOCs in uninfested plants

When infested with Fusarium sp., the cereals Triticum aestivum L. emend. Fiori et Paol. cv. ‘Bombona’, Avena sativa L. cv. ‘Deresz’, and Hordeum vulgare L. cv. ‘Rastik’ can emit volatile organic compounds (VOCs). The VOCs differ both qualitatively and quantitatively from those emitted by non-infested wheat, oat, and barley plants. We detected increased amounts of VOCs released by green leaves (green leaf volatiles – GLVs): (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-hexen-1-ol, (Z)-3-hexen-1-yl acetate, 1-hexyl acetate as well as the other VOCs like (Z)-ocimen, linalol, linaloloxide, benzyl acetate, indole, and β-caryophyllene. The lipoxygenase pathway resulted in the highest release of GLVs in comparison to the other biochemical pathways of volatile production. As a result of Fusarium infestation, the amounts of VOCs varied between tested cereals. We also subjected uninfested wheat, barley, and oat plants to infested wheat plants, and found that these cereals released larger amounts of VOCs compared to control plants. Emitted amounts of VOCs were significantly higher the shorter the distance between uninfested and infested plants.     

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.     

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.     

The C28 aldehyde octacosanal is a morphogenetically active component involved in host plant recognition and infection structure differentiation in the wheat stem rust fungus

Using Langmuir–Schäfer technology, a very thin, homogeneous coating of glass surfaces with leaf epicuticular waxes was achieved, allowing the establishment of a bio-assay for morphogenetically active, hydrophobic surface components triggering infection structure differentiation in a rust fungus. A chloroform extract of wheat leaf epicuticular wax coated onto a flat glass surface induced the formation of appressoria, substomatal vesicles, and infection hyphae in about 50% of the germ tubes of Puccinia graminis f.sp. tritici. No induction was seen on a glass surface coated with an inert wax which confered the same hydrophobicity to the surface as the leaf wax. The chemical composition of the leaf epicuticular wax extract was analysed, and the active ingredient was shown to be the C28-aldehyde, octacosanal. The importance of multiple recognition of a ‘host associated surface pattern’ (HASP) integrating both physical and chemical signaling cues for the crucial differentiation of an appressorium upon recognition of a host stoma is discussed.     

Leaf wax layer may prevent appressorium differentiation but does not influence orientation of the leaf rust fungus Puccinia hordei on Hordeum chilense leaves

The infection process of most rust fungi start with spore germination, directional growth of the germ tube towards a stoma, differentiation of an appressorium over the stoma, and penetration into the substomatal cavity. In the South American wild barley Hordeum chilense Roem. & Schult., wide variation occurs in the degree to which several rust fungal species are able to form appressoria over the stomata. Apparently, features of the plant may hamper early stages of the infection process. Such an early defence is called avoidance. In order to find out how germ tube growth is directed towards stomata, and whether the cuticular wax layer plays a role in this orientated growth and in appressorium differentiation, several orientation and differentiation parameters of Puccinia hordei germ tubes were measured on H. chilense leaves with and without the wax layer. Orientated growth of the germ tubes started upon contact with the epidermal cell junctions. The growth of lateral branches of the germ tube over the first epidermal cell junction that it meets, may help the germ tube to grow along the transverse axis of the leaf. No evidence was found of attraction of the germ tube to stomata. Removal of the cuticular wax layer did not result in loss of germ tube orientation. This suggests that the leaf wax layer has no role in the guidance of germ tubes. On high avoidance accessions, removal of the wax layer allowed appressoria to develop over stomata that would otherwise be overgrown. No effect of the cell widths in stomatal complexes was found on the chance that stomata were overgrown. This suggests that the overgrowth of stomata on H. chilense leaves by P. hordei germ tubes is mainly caused by the wax covering of the stomatal apparatus.     

Cell-substratum adhesive protein involved in surface contact responses of the bean rust fungus

Uredospore germlings of the bean rust fungus Uromyces appendiculatus display two contact-sensitive responses on leaves or certain synthetic surfaces: a specific orientation of germ tubes and an induction of appressoria at leaf stomates or scratched surfaces. Germ tube orientation, nuclear division (used as a marker for appressorium formation) and germling adhesion on scratched “Parafilm” was reduced by the proteolytic enzyme, pronasc E (ED₅₀ = 20–50 μg ml⁻¹), but not by the heat denatured proteolytic enzyme. Forty micrograms of pronase E per millilitre significantly reduced nuclear division of germlings incubated on either a hydrophilic or a hydrophobic surface. Pronase E reduced adhesion regardless of whether the enzyme was applied during growth or after attachment had occurred, although higher concentrations were required after attachment had occurred. The data suggest that extracellular protein is required for germling adhesion to a substratum and that adhesion is required for germ tube orientation and thigmodifferentiation.We partially characterized the composition of the bean rust extracellular material and analyzed for proteins on SDS-polyacrylamide gels. Intact germlings have six predominant extracellular peptides detected by ¹²⁵I labelling. Spent culture fluid contains several different peptides; four of the five major peptides in the spent culture fluid are glycosylated.     

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.     

Mechanism and molecular markers associated with rust resistance in a chickpea interspecific cross (Cicer arietinum × Cicer reticulatum)

A gene that controls resistance to chickpea rust (Uromyces ciceris-arietini) has been identified in a recombinant inbred line (RIL) population derived from an interspecific cross between Cicer arietinum (ILC72) × Cicer reticulatum (Cr5-10), susceptible and resistant to rust, respectively. Both parental lines and all RILs displayed a compatible interaction but differed in the level of infection measured as Disease Severity (DS) and Area Under the Disease Progress Curve (AUDPC). Histological studies of the seedlings of resistant parental Cr5-10 line revealed a reduction in spore germination, appressorium formation, number of haustoria per colony and colony size, with little host cell necrosis, fitting the definition of partial resistance. A Quantitative Trait Locus (QTL) explaining 31% of the total phenotypic variation for DS in seedlings and 81% of the AUDPC in adult plants in the field was located on linkage group 7 of the chickpea genetic map. The AUDPC displayed a bimodal distribution with high frequency of susceptible lines and both the AUDPC and markers showed the same distorted segregation. Consequently, it was hypothesised that a single dominant gene (proposed as Uca1/uca1) controlled resistance to rust in adult plants. This allowed us to locate the gene on the genetic linkage map. Two Sequence Tagged Microsatellite Sites (STMS) markers, TA18 and TA180 (3.9 cM apart) were identified that flank the resistance gene. These findings could be the starting point for a Marker-Assisted Selection (MAS) programme for rust resistance in chickpea.     

Two class III chitin synthases specifically localized in appressoria and haustoria of Puccinia graminis f. sp. tritici

Rust fungi like Puccinia graminis f. sp. tritici are known to change their cell wall properties upon entering the plant tissue. Immunohistochemistry revealed the cellular localization of two class III chitin synthase isoforms in rust mycelia developing on and in the host plant. Isoform IIIa is restricted to fungal infection structures growing on the surface of the plant, such as germ tubes and, predominantly, appressoria. Isoform IIIb is found exclusively in haustoria developed inside the plant. Thus, the rust fungus uses at least two chitin synthase isoforms with specialized functions in the differentiation of infection structures during the biotrophic plant-pathogen-interaction.     

Germination and appressiorial formation by uredospores ofUromyces viciae-fabae exposed to inhibitors of polyamine biosynthesis

An examination was made of the effects of three polyamine biosynthesis inhibitors on germination and appressorium formation by uredospores of the bean rust fungusUromyces viciae-fabea on artificial membranes. The ornithine decarboxylase inhibitor -difluoromethylornithine had no effect on uredospore germination, even when used at 2mM, whereas appressorium formation was reduced by 63% at 0.5 mM and by 99% at 2mM. Methylglyoxal bis(guanylhydrazone), an inhibitor of S-andenosylmethionine decarboxylase, reduced germination when used at 0.025 mM, and at this concentration, appressorium formation was completed prevented. Uredospore germination was unaffected by as much as 3 mM cyclohexylamine, an inhibitor of spermidine esynthase, while appressorium formation was reduced at 1 mM and completely prevented at 3.3 mM. These results support previous suggestions that inhibitors of polyamine biosynthesis exert their main effect on the early stages of fungal development on the leaf surface.Abbreviantions CHA cyclohyxylamine - DFMO -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Induction, Regulation, and Role in Pathogenesis of Appressoria in Monilinia fructicola

ABSTRACT Monilinia fructicola, which causes brown rot in stone fruit, forms appressoria on plant and artificial surfaces. On nectarine, the frequency of appressoria produced by conidial germlings depends to a large degree on the stage of fruit development, with numerous appressoria formed on immature (stage II) nectarine fruit, and no appressoria observed on fully mature fruit (late stage III). On polystyrene surfaces, appressorium formation was increased from <10% of germinated conidia to >95% of germinated conidia when the conidia were washed to remove residual nutrients and self-inhibitors. M. fructicola appressorium formation also appears to be regulated by the topography of the plant surface. On fruit, appressoria formed on stomatal guard cell lips, on the grooves of lateral cells adjacent to stomata or between two epidermal cells, and on the convex surfaces of epidermal cells. Pharmacological effectors indicate that cyclic AMP-, MAP kinase-, and calcium/calmodulin-dependent signaling pathways are involved in the induction and development of appressoria. KN-93, an inhibitor of calmodulin-dependent protein kinase II, did not inhibit conidial germination but did inhibit appressorium formation and brown rot development on flower petals, suggesting that appressoria are required for full symptom development on Prunus spp. petals.     

Green-odour compounds have antifungal activity against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Four green-odour compounds—trans-2-hexenal, cis-3-hexenol, n-hexanal, and cis-3-hexenal—were applied (0.85 μg ml⁻¹ as vapour) to rice plants in laboratory conditions to observe their biological activity against the phytopathogenic fungus Maganporthe oryzae, which causes rice blast disease worldwide. Two compounds, trans-2-hexenal and cis-3-hexenal, showed remarkable disease suppression efficacy (99.7% and 100% suppression, respectively), while n-hexanal had moderate (86.5%) and cis-3-hexenol had weak (20.8%) disease-suppressing effects. Pre-application and post-application of trans-2-hexenal or cis-3-hexenal had slight effects on blast incidence, suggesting that these compounds had direct effects to suppress M. oryzae infection. In fact, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression effect on M. oryzae. Interestingly, these two compounds inhibited appressorium formation at lower concentrations than the growth suppression. Studies on the hypersensitive response (HR)-like reaction and plant β-1,3-glucanase activity in rice plant confirmed that induced resistance was not the major factor involved in the disease suppression mechanism. Results of this study conclusively showed that trans-2-hexenal and cis-3-hexenal possess potent inhibitory activities against the growth and the appressorium formation of M. oryzae and could be used as antifungal agents to significantly reduce M. oryzae infections in rice.     

Development of the infection strategy of the hemibiotrophic plant pathogen,Colletotrichum orbiculare,and plant immunity

The hemibiotrophic fungus Colletotrichum orbiculare forms appressoria as infection structures and primarily establishes biotrophic infection in cucumber epidermal cells. Subsequently, it develops necrotrophic infection. In the pre-invasion stage, morphogenesis of appressoria of C. orbiculare is triggered by signals from the plant surface. We found that C. orbiculare PAG1 (Perish-in-the-Absence-of-GYP1), a component of MOR [morphogenesis-related NDR (nuclear Dbf2-related) kinase network] plays an essential role as a key component of the plant-specific signaling pathway and that hydrolysis of cutin by a spore surface esterase creates a cutin monomer that constitutes a key plant-derived signal. Development of the infection structure of C. orbiculare is strictly regulated by the cell cycle and we found that proper regulation of G1/S progression via two-component GAP genes, consisting of BUB2 (Budding-Uninhibited-by-Benomyl-2) and BFA1 (Byr-Four-Alike-1) is essential for the establishment of successful infection. In the post-invasion stage, the establishment of the biotrophic phase of hemibiotrophic fungi is crucial for successful infection. We found that C. orbiculare WHI2 (WHIsky-2), an Saccharomyces cerevisiae stress regulator homolog, is involved in the phase transition from biotrophy to necrotrophy through TOR (Target of Rapamycin) signaling, and is thus essential for full pathogenesis.     

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.     

普通小麦-华山新麦草易位系9020-17-25-6抗条锈性遗传分析及分子标记

为明确普通小麦-华山新麦草易位系9020-17-25-6的抗条锈病基因及其遗传特点,利用中国条锈菌小种CYR29对9020-17-25-6、铭贤169及其杂交后代F1、F2、F3代进行苗期抗条锈性鉴定及遗传分析,选取48条RGAP引物和491对SSR引物对接种CYR29的F2代群体进行筛选,寻找与抗病基因连锁的分子标记。结果表明:9020-17-25-6对CYR29具有良好的抗条锈性,由1对显性基因独立控制,暂定名为Yr Hua9020。筛选到2个RGAP标记(M1和M2)和位于染色体3AS上的4个SSR标记(Xwmc11、Xwmc532、Xcfd79、Xgwm2)与Yr Hua9020连锁,与目的基因的遗传距离分别为6.9、9.5、17.8、12.2、7.2和17.8 c M。与已定位于3A染色体上的抗条锈病基因的比较研究表明,Yr Hua9020是一个与已知基因不同的新的抗条锈病基因。