Prepenetration Stages of Guignardia psidii in Guava: Effects of Temperature, Wetness Duration and Fruit Age

This study examined the effects of temperature and wetness duration in vitro and in vivo as well as the effects of fruit age on germination and appressoria formation by conidia of Guignardia psidii , the causal agent of black spot disease in guava fruit. The temperatures tested for in vitro and in vivo experiments were 10, 15, 20, 25, 30, 35 and 40°C. The wetness periods studied were 6, 12, 24, 36 and 48 h in vitro and 6, 12 and 24 h in vivo . Fruit 10, 35, 60, 85 and 110-days old were inoculated and maintained at 25°C, with a wetness period of 24 h. Temperature and wetness duration affected the variables evaluated in vitro and in vivo . All variables reached their maximum values at between 25 and 30°C with a wetness duration of 24 h in vivo and 48 h in vitro . These conditions resulted in 31.3% conidia germination, 33.6% appressoria formation and 32.5% appressoria melanization in vitro , and 50.4% conidia germination and 9.5% appressoria formation in vivo . Fruit age also influenced these factors. As fruit age increased, conidia germination and appressoria formation gradually increased. Conidia germination and appressoria formation were 10.8% and 2.3%, respectively, in 10-day-old fruits. In 110-day-old fruits, conidia germination and appressoria formation were 42.5% and 23.2% respectively.     

Epicatechin and catechin may prevent coffee berry disease by inhibition of appressorial melanization of Colletotrichum kahawae

Colletotrichum kahawae is the causal agent of coffee berry disease. Appressorial melanization is essential for the fungal penetration of plant cuticle. Epicatechin is abundant in green coffee berry pericarp. Inoculation of C. kahawae conidial suspension containing 1.2 mg epicatechin or catechin/ml did not affect conidial germination or appressorial formation but appressorial melanization was completely inhibited and infection by the treated conidia was less than 30% of the untreated control. Epicatechin and catechin may, therefore, prevent coffee berry disease by inhibition of the appressorial melanization of C. kahawae.     

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

Melanization of appressoria is critical for the pathogenicity of <Emphasis Type="Italic">Diplocarpon rosae</Emphasis>

Previous studies have shown the role of melanized appressoria in the pathogenicity of various fungi. Diplocarpon rosae is a worldwide outdoor fungal pathogen of rose plants causing black spot disease of rose leaves. To fully understand how this fungus colonizes its host, which is critical for the development of an efficient and sustainable disease management program, we studied the fungal (especially the appressoria) structures of D. rosae in detail at an early stage of infection. Using both microscopic and biochemical analyses, we observed strong melanized appressoria formation localized at the point of D. rosae penetration, which forms the pathogen–plant interface. Treatment of infected plants with melanin biosynthesis inhibitors (MBIs) prevented melanization of D. rosae appressoria and positively correlated with significant reductions in black spot disease symptoms, suggesting that melanization of appressoria might be a critical factor for the pathogenicity of D. rosae. Our findings were confirmed and validated by the lack of melanized appressorial ring formation on an artificial surface and on a D. rosae-non host plant system, Arabidopsis thaliana. Our findings suggest that localized melanization of appressoria is a crucial factor for the pathogenicity of D. rosae and treatment of the fungus with MBIs seems to be a promising disease management alternative for black spot disease of roses.     

Factors influencing spore germination,appressoria formation and disease development inCamellia sinensis byGlomerella cingulata

Factors associated with conidial germination and appressoria formation ofGlomerella cingulata causing the brown blight disease of tea (Camellia sinensis) were studiedin vitro. Spore germination and appressoria formation were optimum at a temperature of 25°C, pH 5.0, 7 h light/day regime and a 24-h incubation period. At a concentration of conidia of 1200/μL 10-d-old culture,G. cingulata exhibited a maximum germination and appressoria formation. A maximum production of lesions was also evident on detached tea leaves at this spore concentration and in diffuse light. Diffusates of a phenolie nature collected from tea varieties susceptible and resistant toG. cingulata inhibited spore germination and appressoria formation. Diffusates from resistant varieties were more fungitoxic than those from susceptible varieties. Some phenolics known to be present in tea leaves, when testedin vitro, exhibited varying degrees of fungitoxicity. Pyrogallol totally inhibited spore germination, while pyrocatechol and phloroglucinol completely inhibited appressoria formation.     

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.     

The Infection of Soybean Leaves by Phakopsora pachyrhizi during Conditions of Discontinuous Wetness

The ability of Phakopsora pachyrhizi to cause infection under conditions of discontinuous wetness was investigated. In in vitro experiments, droplets of a uredospore suspension were deposited onto the surface of polystyrene. After an initial wetting period of either 1, 2 or 4 h, the drops were dried for different time intervals and then the wetness was restored for 11, 10 or 8 h. Germination and appressorium formation were evaluated. In in vivo experiments, soybean plants were inoculated with a uredospore suspension. Leaf wetness was interrupted for 1, 3 or 6 h after initial wetting periods of 1, 2 or 4 h. Then, the wetting was re‐established for 11, 10 or 8 h, respectively. Rust severity was evaluated 14 days after inoculation. The germination of the spores and the formation of the appressoria on the soybean leaves after different periods of wetness were also quantified in vivo by scanning electron microscopy. P. pachyrhizi showed a high infective capacity during short periods of time. An interruption of wetness after 1 h caused average reductions in germination from 56 to 75% and in appressorium formation from 84 to 96%. Rust severity was lower in all of the in vivo treatments with discontinuous wetness when compared to the control plants. Rust severity was zero when the interruption of wetness occurred 4 h after the initial wetting. Wetting interruptions after 1 and 2 h reduced the average rust severity by 83 and 77%, respectively. The germination of the uredospores on the soybean leaves occurred after 2 h of wetness, with a maximum germination appearing after 4 h of wetness. Wetness interruption affected mainly the spores that had initiated the germination.     

Mitochondrial β‐oxidation regulates organellar integrity and is necessary for conidial germination and invasive growth in Magnaporthe oryzae

Fatty acids stored as triglycerides, an important source of cellular energy, are catabolized through β‐oxidation pathways predicted to occur both in peroxisomes and mitochondria in filamentous fungi. Here, we characterize the function of Enoyl‐CoA hydratase Ech1, a mitochondrial β‐oxidation enzyme, in the model phytopathogen Magnaporthe oryzae. Ech1 was found to be essential for conidial germination and viability of older hyphae. Unlike wild‐type Magnaporthe, the ech1Δ failed to utilize C14 fatty acid and was partially impeded in growth on C16 and C18 fatty acids. Surprisingly, loss of β‐oxidation led to significantly altered mitochondrial morphology and integrity with ech1Δ showing predominantly vesicular/punctate mitochondria in contrast to the fused tubular network in wild‐type Magnaporthe. The ech1Δ appressoria were aberrant and displayed reduced melanization. Importantly, we show that the significantly reduced ability of ech1Δ to penetrate the host and establish therein is a direct consequence of enhanced sensitivity of the mutant to oxidative stress, as the defects could be remarkably reversed through exogenous antioxidants. Overall, our comparative analyses reveal that peroxisomal lipid catabolism is essential for appressorial function of host penetration, whereas mitochondrial β‐oxidation primarily contributes to conidial viability and maintenance of redox homeostasis during host colonization by Magnaporthe.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

Effect of temperature, light and host on prepenetration development of Puccinia graminis avenae and Puccinia coronata avenae

The influence of temperature and light on prepenetration development of single and mixed isolates of Puccinia graminis avenae and Puccinia coronata avenae was studied on 0–2% water agar and on leaves of three oat cultivars and on three non-cultivated species of Avena. Germination of uredospores of P. graminis avenae and P. coronata avenae occurred best at 10–30^oC and at 20^oC respectively. The optimum temperature for germ-tube growth and for appressorial formation was 20^oC for both rusts. An inverse relationship was observed between light intensity and prepenetration development with maximal germination of uredospores, germ-tube growth and appressorial formation occurring in darkness. Under optimum conditions maximum percentage germination and appressorium formation of both rusts was attained within 4 and 12 h after inoculation respectively. The proportion of germinated uredospores of crown rust which gave rise to appressoria was about twice that observed for stem rust. No significant differences were observed in prepenetration development between the single and mixed race inocula of the two rusts. Although germination of uredospores was significantly greater on water agar than on oat leaves, there were no significant differences in prepenetration development of the rusts on the various oat cultivars and species examined. Consequently, the data failed to indicate the presence of resistance mechanisms operating during the prepenetration phase of the infection process on the cultivars and species examined.     

An adaptor protein BmSte50 interacts with BmSte11 MAPKKK and is involved in host infection,conidiation, melanization,and sexual development in Bipolaris maydis

A mitogen-activated protein kinase (MAPK) signaling pathway regulates specialized cellular responses to external stimuli. In Bipolaris maydis, a Chk1 MAPK orthologous to Fus3/Kss1 MAPKs of Saccharomyces cerevisiae is known to regulate various developmental processes, including the formation of appressoria. However, upstream factors that regulate the Chk1 cascade have not been well clarified. In this study, we identified and characterized the BmSte50 gene, an ortholog of the yeast Ste50 in B. maydis. Our yeast two-hybrid assay indicated that BmSte50 interacts with a MAPK kinase kinase BmSte11, a component of the Chk1 cascade. ΔBmSte50 strains exhibited a loss of pathogenicity due to a lack of appressorial formation. The mutants also showed a reduction in melanization, conidial production, and aerial-mycelial and sexual development. Such phenotypes of the mutants were consistent with those of the Chk1 cascade gene mutants previously reported. In addition, ΔBmSte50 strains indicated lower conidial germination efficiency than the wild type. Notably, a significant number of ΔBmSte50 conidia could be germinated, while the Chk1 cascade gene mutants were reported to lack conidial germination ability. Our results suggested that BmSte50 may act as an adaptor protein for the Chk1 cascade and is involved in the regulation of various cellular processes.     

Temperatures during flower bud development affect pollen germination,self‐incompatibility reaction and early fruit development of clementine (Citrus clementina Hort. ex Tan.)

• One of the key environmental factors affecting plant reproductive systems is temperature. Characterising such effects is especially relevant for some commercially important genera such as Citrus. In this genus, failure of fertilisation results in parthenocarpic fruit development and seedlessness, which is a much‐prized character. Here, we characterise the effects of temperature on flower and ovary development, and on pollen–pistil interactions in ‘Comune’ clementine (Citrus clementina Hort. ex Tan.).
• We examine flower bud development, in vitro pollen germination and pollen–pistil interaction at different temperatures (15, 20, 25 or 30 °C). These temperatures span the range from ‘cold’ to ‘hot’ weather during the flowering season in many citrus‐growing regions.
• Temperature had a strong effect on flower and ovary development, pollen germination, and pollen tube growth kinetics. In particular, parthenocarpic fruit development (indicated by juice vesicle growth) was initiated early if flowers were exposed to warmer temperatures during anthesis.
• Exposure to different temperatures during flower bud development also alters expression of the self‐incompatibility reaction. This affects the point in the pistil at which pollen tube growth is arrested and confirms the role of sub‐ and supra‐optimal temperatures in determining the numbers of pollen tubes reaching the ovary.

     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Inhibition of melanin biosynthesis by cerulenin in appressoria ofColletotrichum lagenarium

Cerulenin [(2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a polyketide synthesis inhibitor, inhibited appressorial pigmentation ofColletotrichum lagenarium at concentrations higher than 10 μg/ml. The inhibitory concentrations of cerulenin inhibited neither spore germination nor appressorium formation but did inhibit penetration of nitrocellulose membranes by penetrating hyphae from appressoria. The colorless appressoria germinated laterally on nitrocellulose membranes and rarely penetrated them. In the presence of cerulenin, treatment with scytalone, a melanin precursor for this fungus, restored appressorial pigmentation and also penetration by appressoria of nitrocellulose membranes. In Czapek liquid medium, mutant 8015 which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthetic pathway produced scytalone after 9 days of incubation when hyphal growth reached the maximum level. Application of 10 μg/ml of cerulenin at 9 days of incubation inhibited further production of scytalone by mutant 8015. From these results, it is concluded that appressorial melanin ofC. lagenarium was synthesizedvia the polyketide pathway.     

Effect of Light, Temperature and Substrate during Spore Formation on the Germinability of Conidia of Colletotrichum falcatum

This paper presents results on the effect of light, temperature and substrate during spore formation on the germinability of conidia in Colletotrichum falcatum. Light seems to have no effect on the germination of conidia unless the cultures were exposed to a high intensity of light during sporulation, in which case the spores showed a reduced germination and an increased appressoria formation. Conidia produced at temperatures higher than the optimum showed better germination and less appressoria formation than the spores produced at the temperature optimum for the growth and sporulation of the fungus. A similar increase in germination was also observed in conidia obtained from inoculated sugarcane leaves as compared to those produced on culture media. The light type virulent isolates of C. falcatum showed greater sensitivity to all these treatments than the dark type weakly pathogenic isolates.     

The influence of temperature and photoperiod on the reproductive diapause and cold tolerance of spotted‐wing drosophila,Drosophila suzukii

Knowledge regarding the reproductive status of spotted‐wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is of critical importance in predicting potential infestations of this invasive pest, as eggs are laid in ripe or ripening fruit of several commercially important small‐fruit crops. Token environmental stimuli for the induction of reproductive diapause and improved cold tolerance were identified for D. suzukii. Diapause induction was evaluated by assessing, via dissection, the number of mature eggs carried by field‐captured D. suzukii and laboratory‐reared D. suzukii held under various temperature and photoperiod regimes. Egg load decreased over time in females trapped from July to December at sites in Benton County, OR, and Ontario County, NY, both USA, and reached zero eggs by December at all sites. Photoperiods below 14 h of day length led to reduced egg maturation in laboratory‐reared flies held at moderate temperatures (15 or 20 °C). Whereas very few mature eggs were found in females held at 10 °C under short‐ or long‐day photoperiods for several weeks after eclosion, a spontaneous return to ovarian maturity was observed in short‐day‐entrained females after 7 weeks. Diapause termination was investigated by evaluating fecundity in diapausing females returned to optimal environmental conditions. Whereas long‐day‐entrained flies began producing offspring immediately upon return to optimal conditions, short‐day‐entrained flies returned after 1 and 6 weeks at 10 °C were slower to produce offspring than colony flies or short‐day‐entrained flies returned after 7 weeks. Cold tolerance was evaluated by observing chill coma recovery rates after 24 h exposure to ?1 °C. Cold‐acclimated (diapausing) females recovered from chill coma faster than cold‐hardened or unacclimated females.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Morpho‐histological and ultra architectural changes during early development of endangered golden mahseer Tor putitora

Ultrastructural and histological changes in the embryonic and larval surface during ontogenesis of the endangered golden mahseer Tor putitora is studied here for the first time. Embryonic development was completed 91–92 h after fertilization at an ambient temperature of 23° ± 1° C (mean ± s.d. ). The gastrula stage was characterized by presence of the Kupffer's vesicle, notochord, ectoderm and endoderm cells. Primordial germ cells were clearly identifiable from c. 55 h post‐fertilization at the organogenesis stage. Mean total length of newly hatched larvae was 7·0 ± 0·5 mm. Scanning electron microscopy of newly hatched larvae demonstrated vitelline arteries, microridged epithelial cells and mucous gland openings over much of the body surface. Eye, oral cavity, pharyngeal arches, heart, intestinal loop, prosencephalon, cephalic vesicle and nasal epithelium were clearly distinguished in 3 day old hatched individuals. In 6 day old individuals, caudal‐fin rays and internal organs were evident. The dorsal fin became prominent at this stage and larvae began swimming at the surface. The reserved yolk material was totally absorbed 8–11 days after hatching and larvae began feeding exogenously. Tor putitora exhibited a longer early developmental period than other cyprinids reared at similar temperatures.