Conidia of the tomato powdery mildew <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> initiate germ tubes at a predetermined site

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector—this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.     

Attachment and germination of Entomophaga maimaiga conidia on host and non-host larval cuticle

The lepidopteran-specific fungal pathogen Entomophaga maimaiga is highly virulent against Lymantria dispar (gypsy moth) larvae, and other members of the family Lymantriidae. Numerous species in the subfamily Cuculliinae (Family Noctuidae) are not susceptible to E. maimaiga due to the inability of this fungus to penetrate the larval cuticle. Conidial attachment and germination were compared among five cuculliine species and L. dispar using bioassays and scanning electron microscopy. Although conidia were showered evenly across larvae during bioassays, on L. dispar conidia were most abundant on segments, where they adhered well to the cuticle and germinated at high percentages. Conidia on cuculliine cuticles were predominantly found in large, loose aggregations in intersegmental areas. Few conidia on cuculliine cuticle germinated and scanning electron microscopy revealed a thick film of mucous enveloping conidia. We hypothesize that the conidia on cuculliines become coated by this film and were only loosely attached to the larval cuticle. No such film was seen on L. dispar larvae where individual conidia appeared well attached. On L. dispar larvae many conidia also adhered to setae. To determine if hydrophobicity affected the ability of E. maimaiga conidia to attach and germinate on a substrate, a goniometer was used to determine relative hydrophobicity of larval cuticles. L. dispar cuticle was more hydrophobic than cuculliine cuticle, suggesting that a high level of hydrophobicity could be a required characteristic for hosts. Cuticles from four cuculliine species and L. dispar were sequentially extracted using hexane, chloroform, and methanol. Conidia were showered onto glass slides coated with the different extracts and germination was quantified. Methanol extracts of cuculliine cuticle consistently decreased germination, compared to all extracts of L. dispar cuticle. For all L. dispar extracts, the majority of conidia produced germ tubes, which is a normal prerequisite for cuticular penetration. For the cuculliines, conidia exposed to hexane and chloroform extracts produced secondary conidia as did all controls, but the conidia exposed to cuculliine methanol extracts that germinated produced germ tubes. These studies demonstrated that a range of factors act in concert to prevent E. maimaiga infection of the cuculliine species investigated.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.     

Scanning Electron Microscopy of the Infection Process of Cercospora henningsii on Cassava Leaves

Germination, penetration and sporulation of Cercospora henningsii (Allesch.) on cassava leaves were studied by scanning electron microscopy. Conidia started to germinate 9 h postinoculation producing one to two germ tubes. The germ tubes entered the leaf tissue through the abaxial surface by direct penetration of the epidermis without forming appressoria. The cassava leaf is characterized by its papillose epidermis on the abaxial surface. The penetrations occurred at smooth areas of the leaf epidermis between the papillae. The germ tubes did not enter stomata even when they passed over stomatal openings. Leaf spots started to appear 9 days after the inoculation (dpi), and the emergence of conidia occurred 14 dpi. The symptoms appeared first on the abaxial leaf surface, followed 2 days later on the adaxial. Conidia emerged in clusters through ruptured epidermis on both sides of the leaves. Conidia emerged also through the epidermal papillae and the leaf veins. Even though small groups of conidia emerged through stomata also, emergence through stomata appeared to be random rather than a preferred route. Each conidium was born on a short conidiophore with a swollen base.     

Detection of protein and carbohydrate in the extracellular matrix released byCochliobolus heterostrophus during germination

The extracellular matrix (ECM) ofCochliobolus heterostrophus (anamorph:Bipolaris maydis) was made visible by gold/silver and FITC-lectin staining at different stages of germ tube development. A proteinaceous material was released from conidia as germ tubes began to emerge and continued to be released from the germ tube tip throughout elongation. A material that did not stain for protein was observed to surround germ tubes upon their elongation. At later stages of maturation, germ tubes were surrounded by a sheath of proteinaceous material. After 15 h of incubation, staining with the FITC-labeled Concanavalin A revealed that a carbohydrate material surrounded and extended between hyphae. The ECM extract was separated into two fractions which were shown by SDS-PAGE and HPLC analyses to consist of proteins and carbohydrates. The results demonstrate that the composition and physical structure of the ECM change over time. Thus, the ECM is not a static material. Rather, the components of the ECM appear to be laid down at different stages of fungal morphogenesis, possibly related to germ tube emergence, elongation, and maturation.     

Adhesion of Nongerminated Botrytis cinerea Conidia to Several Substrata

Conidia of the plant pathogenic fungus Botrytis cinerea adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of B. cinerea depends, at least in part, on hydrophobic interactions between the conidia and substratum.     

Evidence that the cAMP pathway controls emergence of both primary and appressorial germ tubes of barley powdery mildew

Development of conidia of barley powdery mildew involves the formation of a primary germ tube (PGT), an appressorial germ tube (AGT), and an appressorium. Previously, it was found that cyclic AMP (cAMP) was involved in these developmental processes. Comparison of development on the host surface with two types of cellulose membrane revealed that frequency of PGT emergence was surface independent. On one type of cellulose, where the frequencies of both AGT and appressorial differentiation were similar to that on the host surface, cAMP levels and protein kinase A (PKA) activities had a biphasic pattern with peaks at 15 min and 4 h after inoculation (prior to PGT and AGT emergence, respectively). The effect of manipulating cAMP levels was tested on another type of cellulose membrane, which stimulated a lower degree of AGT and appressorial formation than the host surface. Cholera toxin and forskolin, activators of adenylyl cyclase, significantly increased PGT emergence, but cAMP did not. Cholera toxin, forskolin, and cAMP increased the frequency of AGT and appressorial formation, but in a time-dependent manner.     

“Pollination” of a fungus by a fly

Summary The mechanism resulting in fertilization of Epichloë typhina, a heterothallic ascomycete that is an endophytic pathogen of grasses, has now been discovered. Conidia of one mating type are produced in stromata and are then transferred by insects to individuals of the opposite mating type. One insect, Phorbia phrenione, is a particularly important vector of conidia. Once conidia of the opposite mating type have been transferred to a stroma, the life cycle continues with the formation of perithecia.     

Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.     

Deposition and germination of conidia of the entomopathogen Entomophaga maimaiga infecting larvae of gypsy moth,Lymantria dispar

Germination of conidia of Entomophaga maimaiga, an important fungal pathogen of gypsy moth, Lymantria dispar, was investigated on water agar and larval cuticle at varying densities. Percent germination was positively associated with conidial density on water agar but not on larval cuticle. When conidia were showered onto water agar, the rate of germination was much slower than on the cuticle of L. dispar larvae. From the same conidial showers, the resulting conidial densities on water agar were much higher than those on larval cuticle in part because many conidia adhered to setae and did not reach the cuticle. A second factor influencing conidial densities on larval cuticle was the location conidia occurred on larvae. Few conidia were found on the flexible intersegmental membranes in comparison with the areas of more rigid cuticle, presumably because conidia were physically dislodged from intersegmental membranes when larvae moved. Conidia were also exposed to heightened CO(2) to evaluate whether this might influence germination. When conidia on water agar were exposed to heightened CO(2) levels, germinating conidia primarily formed germ tubes while most conidia exposed to ambient CO(2) rapidly formed secondary conidia.     

几种虫生真菌附着胞的荧光显微及扫描电镜观察

本研究通过对细脚拟青霉、蝉拟青霉、玫烟色拟青霉、金龟子绿僵菌和莱氏野村菌在疏水表面产生附着胞的荧光显微镜观察和扫描电镜观察,明确五种虫生真菌均可产生附着胞,细脚拟青霉和金龟子绿僵菌产生单附着胞和复合附着胞两种形态,均呈椭圆至长椭圆形,如遇到不适合浸染的部位,则重新产生芽管向前延伸直至找到适合入侵的部位。蝉拟青霉分生孢子多在顶端发芽成人字形,末端形成椭圆形附着胞,该拟青霉再生附着胞能力强。金龟子绿僵菌和莱氏野村菌附着胞表面有较厚的粘液层,而三种拟青霉附着胞表面均未见有粘液层。     

Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates

Solar ultraviolet radiation (UV-A and UV-B) is a major factor in failure of programs using the insect pathogenic fungus Metarhizium anisopliae as a biological control agent. Studies were conducted to determine if growth conditions, viz. artificial (agar media or rice grain) or natural (infected insects) substrates for conidial production affect two traits that directly influence performance of conidia after field application: tolerance to UV-B radiation and conidial germination speed. Conidia of two isolates (ARSEF 23 and ARSEF 2575) of M. anisopliae var. anisopliae produced on potato dextrose agar plus yeast extract (PDAY) or on fungus-killed larvae of two insect species, Galleria mellonella and Zophobas morio, were inactivated by exposure to UV-B radiation. Conidia of both isolates when produced on insect cadavers were significantly more sensitive to UV-B radiation than conidia produced on PDAY. Also, conidia from insect cadavers germinated slower than those from PDAY cultures. A comparison of conidia from artificial substrates showed that conidia produced on Czapek's and Emerson's YpSs agar media or rice grains had higher tolerance to UV-B radiation and germinated faster than conidia raised on PDA and PDAY. Accordingly, the growth substrate and nutritional environment in which conidia are produced influences M. anisopliae conidial UV-B tolerance and speed of germination; and manipulation of these variables could be used to obtain conidia with increased tolerance to UV-B radiation and shorter germination times.     

Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

Abstract

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.     

Effects of Culture Age, Washing and Storage Conditions on Desiccation Tolerance of Colletotrichum truncatum Conidia

Colletotrichum truncatum conidia produced from a one week-old culture in a liquid semi-defined medium with a C:N ratio of 5:1 were more tolerant of desiccation than those harvested from two or three week-old cultures. Conidia washed with 20% (w/v) sucrose germinated better than unwashed conidia or those washed in 10% (w/v) sucrose, 10 and 20% (w/v) glucose or fructose, 0.1% (w/v) soluble starch, 0.9% (w/v) NaCl or deionized water. Washing with sucrose (20% w/v) also resulted in significantly longer germ tubes than those produced by unwashed conidia or conidia washed with deionized water or NaCl (0.9% w/v). Conidia washed twice in sucrose showed greater desiccation tolerance during storage at 15% relative humidity (RH) and 15°C than at 30% RH and 15 or 25°C or at 15% RH and 25, 5 or -10°C.     

Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

Generality of the prerequisite of conidium attachment to a hydrophobic substratum as a signal for germination among Phyllosticta species

It has been shown that conidia of Phyllosticta ampelicida require attachment to a substratum to initiate germination. Furthermore this attachment occurs only on hydrophobic surfaces. This study was initiated to ascertain the breadth of this phenomenon among other species of the genus Phyllosticta. We tested 23 isolates of Phyllosticta representing at least 14 named species. These isolates were collected from North America, Asia and Africa. For 22 of the 23 isolates tested spore attachment occurred at a rate of 60-100% on hydrophobic polystyrene but at 0-5% on hydrophilic polystyrene. The one exception to the preference for a hydrophobic substratum for attachment was an unnamed species of Phyllosticta from Rhus glauca that attached less than 10% on either surface. A similar response was observed when assaying germination and appressorium formation for 17 isolates. Germination and appressorium formation for these isolates proceeded on hydrophobic polystyrene but not on nutrient agar, which is hydrophilic. In five of the tested isolates germination was high on both hydrophobic polystyrene and hydrophilic nutrient media. The isolate from Rhus glauca did not germinate appreciably on either surface. Taken together these results suggest that the requirement for conidium contact/attachment to trigger germination is pervasive to the genus Phyllosticta.     

Appressorium formation and nuclear division in Colletotrichum truncatum

Conidia of the soybean anthracnose fungus, Colletotrichum truncatum differentiate to form appressoria required for host invasion when the germ tube touches a hard surface. This thigmotrophic stimulus appears to be translated by the fungus during the second round of nuclear division. Inhibiting the second round of DNA synthesis by fluorodeoxyuridine or hydroxyurea blocked appearance of appressoria but not emergence of the germ tube. DNA synthesis and mitosis resumed upon removal of FUdR but only mycelia formed, and infection structures did not appear. In addition, actinomycin D reversibly blocked development of appressoria and synthesis of polyadenylate, but nuclear division was not affected. The data suggest that anthracnose conidia produce appressoria in response to germ tube contact by altering the messenger program of its germ tube nucleus. This study has also shown that mitochondrial DNA had an unusual bimodal distribution in CsCl at 1.690 and 1.719 g/cm³, respectively.Non-Standard Abbreviations FUdR 5-fluorodeoxyuridine - polyA polyadenylic acid     

Consecutive monitoring of lifelong production of conidia by individual conidiophores of Blumeria graminis f. sp. hordei on barley leaves by digital microscopic techniques with electrostatic micromanipulation

Conidial formation and secession by living conidiophores of Blumeria graminis f. sp. hordei on barley leaves were consecutively monitored using a high-fidelity digital microscopic technique combined with electrostatic micromanipulation to trap the released conidia. Conidial chains formed on conidiophores through a series of septum-mediated division and growth of generative cells. Apical conidial cells on the conidiophores were abstricted after the conidial chains developed ten conidial cells. The conidia were electrically conductive, and a positive charge was induced in the cells by a negatively polarized insulator probe (ebonite). The electrostatic force between the conidia and the insulator was used to attract the abstricted conidia from the conidiophores on leaves. This conidium movement from the targeted conidiophore to the rod was directly viewed under the digital microscope, and the length of the interval between conidial septation and secession, the total number of the conidia produced by a single conidiophore, and the modes of conidiogenesis were clarified. During the stage of conidial secession, the generative cells pushed new conidial cells upwards by repeated division and growth. The successive release of two apical conidia was synchronized with the successive septation and growth of a generative cell. The release ceased after 4-5 conidia were released without division and growth of the generative cell. Thus, the life of an individual conidiophore (from the erection of the conidiophore to the release of the final conidium) was shown to be 107 h and to produce an average of 33 conidia. To our knowledge, this is the first report on the direct estimation of life-long conidial production by a powdery mildew on host leaves.     

Variability in susceptibility to simulated sunlight of conidia among isolates of entomopathogenic Hyphomycetes

The influence of simulated sunlight on survival of conidia of 4 species of entomopathogenic Hyphomycetes was investigated. Conidia from 65 isolates ofBeauveria bassiana, 23 ofMetarhizium anisopliae, 14 ofMetarhizium flavoviride and 33 isolates ofPaecilomyces fumosoroseus were irradiated by artificial sunlight (295 to 1,100 nm at an ultraviolet-B irradiance of 0.3 W m^–2) for 0, 1, 2, 4 and 8 h. Survival was estimated by comparing the number of colony forming units (CFU) produced by conidia exposed to irradiation to the number of CFUs produced by an unexposed control. Survival decreased with increased exposure to simulated sunlight; exposure for 2 h or more was detrimental to all isolates tested. Overall, isolates ofM. flavoviride were the most resistant to irradiation followed byB. bassiana andM. anisopliae. Conidia ofP. fumosoroseus were most susceptible. In addition to the large interspecies differences in susceptibility to irradiation, there was also an intraspecies variation indicating that strain selection to irradiation tolerance may be important in the development of microbial control agents where increased persistence in an insolated environment is desirable.Abbreviations CFU Colony forming units - UV-B ultraviolet radiation-B