Evaluation of an Inoculum Production and Delivery Technique for Valdensinia heterodoxa, a Potential Biological Control Agent for Salal

Inoculum production of Valdensinia heterodoxa Peyr. (isolate PFC 3027), a foliar pathogen of salal (Gaultheria shallon Pursh.), was evaluated on various solid substrates. Incubation on salal leaf pieces resulted in the greatest number of discharged conidia, which is deemed a suitable indicator for sporulation and inoculum dispersal of V. heterodoxa. Subsequently, V. heterodoxa inoculum of two isolates (PFC 2761 and PFC 3027) was produced on salal leaf pieces using four different growth periods (0, 4, 8, or 12 days) and three different cold storage treatments (0, 4, or 8 weeks) following harvest of colonised leaf pieces. Overall, the maximum number of discharged conidia was obtained with a growth period of 4 or 8 days, whereas the effect of a cold storage treatment was inconclusive between the fungal isolates and various growth periods. Conidia discharge from salal leaf piece inoculum of V. heterodoxa (PFC 2761 and PFC 3027) applied on pot soil level resulted in 8-41% leaf damage of intact salal plants. Results from this study indicate the potential of salal plant tissue as both a growth substrate and a suitable delivery technique for V. heterodoxa to control salal.     

Effects of culture media, temperature, pH, and light on growth, sporulation, germination, and bioherbicidal efficacy of Phoma exigua, a potential biological control agent for salal (Gaultheria shallon)

In order to evaluate the potential use of Phoma exigua isolate PFC 2705 (PFC2705) as a biological control agent for salal (Gaultheria shallon), effect of cultural and environmental parameters on growth, conidia production, and pathogenicity of P. exigua were characterized in studies conducted under laboratory and greenhouse conditions. Within a range of 5-30°C, the optimum growth and germination temperature range was 20-25°C. The effect of pH on mycelial growth and conidial germination was not significant from pH 5 to 10. Fluorescent light significantly enhanced sporulation of the fungus on most agar media tested, yet was not necessary for growth. The type of culture media significantly affected mycelium growth, sporulation, and conidia germination. Age of mycelia used as inoculum affected the disease severity on salal. PFC2705 suppressed the growth of mature salal plant by inciting lesions on leaves, branch tips, and axillary buds and caused 56% death of the total biomass above ground. Characteristics such as easy inoculum production, wide range of growth environments, and high infectivity on salal increased the potential of P. exigua as a biocontrol agent for management of salal.     

Effect of relative humidity and origin of isolates of Neozygites tanajoae (Zygomycetes: Entomophthorales) on production of conidia from cassava green mite, Mononychellus tanajoa (Acari: Tetranychidae), cadavers

Neozygites tanajoae is a very specialized fungus pathogenic to the cassava green mite (CGM), Mononychellus tanajoa, an important cassava pest introduced to Africa from the Neotropics. Conidial discharge from cadavers of CGM that died from infections with 14 isolates of N. tanajoae collected from diverse climates of Brazil was quantified to help select potential candidate strains for introduction to Africa. Studies aimed to identify isolates with lower requirements for relative humidity for sporulation and isolates that discharged more conidia during short periods of moisture. At 96 ± 0.5% RH, production of conidia was variable and even isolates from the Brazilian semi-arid region, e.g., Petrolina and Itaberaba, produced few conidia. Significant differences in the numbers of conidia produced by diverse Brazilian isolates were observed after 6, 9 and 12 h at 100% RH. At 100% RH, production of primary conidia increased considerably from an average of 57 ± 4 conidia at 6 h to 509 ± 37 conidia at 12 h. The isolate sporulating least (BIN21) discharged only 45.7% of the number of conidia produced by isolate BIN1, one of the isolates producing the most spores. Results from this study demonstrate that differences in production of conidia among isolates should be considered when selecting Neozygites isolates for new biological control introductions.     

Substrate influence on physiology and virulence of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> acting on larvae and adults of <Emphasis Type="Italic">Tenebrio molitor</Emphasis>

Two isolates of Beauveria bassiana, wild type (wt) and its mutant type (mt) were compared in terms of growth patterns on culture plates containing media based on wheat bran, grasshopper exoskeletons, colloidal chitin or Sabouraud-dextrose agar (SDA). Germination for the mt isolate was up to 33% faster in all media. Influence of media on virulence was determined against larvae and adults of Tenebrio molitor. Mortality higher than 90% was reached for adults after 6 days using conidia from all media. For larvae, a mortality of 80% was reached after 11 days with conidia collected from SDA medium and between 15 and 35% with conidia from other media. In SDA medium, conidial yield was almost ten times higher for the mt isolate compared to the wt isolate; however, virulence traits were similar against either larvae or adults. These results may influence commercial preparations of entomopathogenic fungi based on conidia.     

A new species of <Emphasis Type="Italic">Discostroma</Emphasis> and its anamorph <Emphasis Type="Italic">Seimatosporium</Emphasis> with two morphological types of conidia,isolated from the stems of <Emphasis Type="Italic">Paeonia suffruticosa</Emphasis>

Conidia of two morphologically different types, one with a basal appendage only and the other with appendage at both ends, were isolated from the stems of Paeonia suffruticosa. Single conidial isolates of both types of conidia yield identical colonies, which then produced both types of conidia on agar media depending on temperature, thus showing that both types of conidia belong to the same fungus. Seimatosporium botan is described based on its morphological characteristics. The teleomorph of the fungus was first found on sterilized P. suffruticosa stems placed on water agar, when grown at 5°C for 2 months in 12-h photoperiod. Discostroma botan is described for this fungus. The teleomorph is also found on the same host in the field.     

Enhancement of sporulation in species of Bipolaris, Curvularia, Drechslera, and Exserohilum by growth on cellulose-containing substrates

Nine species of Bipolaris, Curvularia, Drechslera, and Exserohilum were compared for sporulation on agar media and for enhancement of sporulation by growth on four cellulose-containing substrates (index card, filter paper, cheesecloth, cotton fabric). On two natural and one synthetic agar media, sporulation varied from profuse to nonexistent among three isolates of each species. Growth of all species on cellulose substrates resulted in large and significant increases in sporulation. Growth on index card pieces often provided the greatest increases, but no single substrate was superior for all species, and significant substrate × isolate interactions were observed within species. Overlay of filter paper onto whole colonies in agar plates resulted in 2 to 18-fold increases in sporulation for eight of nine species and production of spores in sufficient quantity for most experimental purposes. Overlay of soil dilution plates with filter paper to promote sporulation of colonies enabled detection of B. spicifera, B. hawaiiensis, C. lunata, and E. rostratum at relatively low population levels (≤1.3 × 10³ colony-forming units per gram of soil) in samples of a naturally infested soil. Results indicate that enhancement of sporulation by growth of species of Bipolaris, Curvularia, Drechslera, and Exserohilum on cellulose substrates may facilitate (i) their identification in culture, (ii) production of spores at relatively high concentrations, and (iii) detection and enumeration of these fungi in soil.     

Susceptibility of the cattle tick Boophilus microplus (Acari: Ixodidae) to isolates of the fungus Beauveria bassiana

The susceptibility of the tick Boophilus microplus to Beauveria bassiana was evaluated by inoculating eggs, larvae and engorged females of the tick with five fungal isolates at concentrations of 10⁶, 10⁷ and 10⁸ conidia/ml. Tick eggs (0.25 g) were immersed in 1 ml of a suspension of the different conidial concentrations for 1 min. Similar exposure was performed by immersion of 2000 larvae and homogeneous groups of nine engorged females in 2 and 20 ml of conidial suspension, respectively. Treated eggs, larvae and adults were placed in an incubator at 27 ± 1 °C and relative humidity above 80% for evaluation of the fungal action. All fungal isolates applied at all conidial concentrations reduced the hatching rate of larvae from treated eggs by 1.36–65.58% and increased the mortality rate of inoculated larvae by 0.8–70.49%. In the bioassay with engorged females, oviposition period was reduced by 9.69–47.80%, egg mass weight by 4.71–53.87%, estimated reproduction by 8.3–60.62%, egg production index by 5.03–54.20%, percent larval hatching by 0.27–13.96%, and the mortality rate of treated females was increased by 96.60–100%. The reduction of the estimated reproduction obtained for the treated groups ranged from 8.37 to 64.52%. The sporulation of the pathogen on dead females ranged from 3.70 to 88.88% depending on the isolate and concentration used. Isolates AM 09, CB 7 and JAB 07 were the most effective and effectiveness increased with increasing concentrations of conidia in the suspensions.     

环境因素对香蕉叶斑病菌喙突脐蠕孢生长和产孢的影响

为进一步明确香蕉叶斑病菌喙突脐蠕孢的生物学特性,调查了部分环境因素(碳氮源、植物成分培养基)对供试菌株(CLER09、D087和JL05)的营养生长和产孢的影响。结果显示,分生孢子主要先从基部萌芽,在28℃下约培养10h后出现两端萌芽;供试菌株对测试的20种碳源及26种氮源显示相似的生长反应,均可利用除菊糖外的测试碳源及氮源进行营养生长和产孢;测试的6种植物成分培养基对供试菌株的营养生长效果皆优于PDA培养基;除燕麦培养基外,其余5种培养基对菌株CLER09和D087的产孢作用均优于PDA培养基;测试的6种培养基对菌株JL05的产孢作用均优于PDA培养基。不同植物成分培养基对该菌分生孢子形态影响较大,以米糠、象草培养基对菌株D087和JL05的分生孢子长度和玉米粉培养基对菌株CLER09的分生孢子宽度的增长效果最为明显。     

环境因子对努利虫疠霉产孢和孢子萌发的影响

探讨努利虫疠霉Pandoranouryi(Remaudi埁re&Hennebert)H櫣mber在田间蚜虫种群中发生与流行的规律,研究外界环境因子对感菌虫尸产孢和孢子萌发的影响。结果表明,处于水琼脂培养平板上感染努利虫疠霉的桃蚜虫尸在8~25℃的温度范围内均能产生大量的初生分生孢子,在30℃下,仅弹射极少量孢子。8℃下,孢子弹射可以持续120h,当温度高于15℃,大部分的孢子会在48h内完成弹射。相对湿度小于95%,虫尸停止产孢。20℃下,光照条件不会影响虫尸弹射孢子的总量。在8℃和30℃时,24h后处于水琼脂培养平板上的孢子萌发率分别为45.23%和61.74%,显著低于15~25℃温度范围内的孢子萌发率(95%以上)。处于叶片上的真菌孢子,当相对湿度大于74%时出现萌发,但在盖玻片的表面,当湿度低于100%时未发现孢子萌发。     

Factors affecting sporulation and germination of Pandora nouryi (Entomophthorales: Entomophthoraecae), a pathogen of Myzus persicae (Homoptera: Aphididae)

Pandora nouryi discharged large numbers of primary conidia between 8 and 25°C from cadavers on the surface of water-agar. At 8°C conidial discharge lasted for 120 h, but most conidia were produced within 48 h when temperature was >15°C. Saturated humidity alone was not enough to allow for sporulation to occur freely and where RH?<?95%, no conidia were discharged. Light did not affect the pattern of conidial production nor the total number of conidia. Germination percentages of conidia on the surface of water-agar were 40 and 66% at 8 and 30°C, respectively, and were significantly lower than that at 15–25°C where germination was >95%. Conidia on leaves germinated well when RH?>?74%, while no germination occurred when RH?<?100% on cover slips. All eight insecticides tested entirely inhibited conidial germination at recommended doses (R), in particular, both the organophosphorus pesticides Lorsben (chlorpyrifos) and the organochlorine pesticides Thiodan (endosulfan) completely inhibited conidial germination even at 0.2R dose.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Materials for the fungus flora of Japan (56)Mariannaea camptospora andM. elegans var.punicea from Japan

One additional species and a variety ofMariannaea, M. camptospora andM. elegans var.punicea, were recorded for the first time in Japan.Mariannaea camptospora formed two types of conidiophores. One type was characterized by simple verticillate phialides sometimes with punctuate walls at the base, producing long oblique conidial chains, and symmetrical spindle-shaped conidia. The other type was characterized by more crowded and shorter phialides with small conidial droplets and hemispherical to concave smaller conidia.Mariannaea elegans var.punicea was characterized by distinct red purple pigmentation in agar media. (55): Udagawa, S. and Uchiyama, S., Mycoscience41: 263–267, 2000.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

Production of zearalenone,moniliformin and trichothecenes in intact sugar beets under laboratory conditions

Five toxigenic isolates of Fusarium species were tested for the production of zearalenone, moniliformin and trichothecenes (deoxynivalenol, 15-acetyldeoxynivalenol, T-2, HT-2 and neosolaniol) when grown on solid sugar beet slices in the laboratory for thirty days. The isolates were also grown on a solid rice medium for comparison. High zearalenone and trichothecene-producing isolates originally obtained from corn and corn-based feedstuff were compared with isolates obtained from sugar beets. One moniliformin-producing isolate from wheat was included in the study. With the exception of moniliformin, all toxins were produced on both substrates; however, the rice medium yielded the greater concentrations except for HT-2 which was produced on sugar beets in equal or greater concentrations. Zearalenone production on rice reached 729–1943 gmg/g whereas on sugar beet it reached 72–193 gmg/g. The moniliformin-producing isolate grew well on both substrates; however, moniliformin was produced only on the rice substrate. This study demonstrates for the first time that Fusarium species can produce both zearalenone and the trichothecenes on a sugar beet substrate.     

Optimising sporulation and virulence in Drechslera avenacea

Studies were conducted on agar media to optimise sporulation of Drechslera avenacea, a fungal pathogen being evaluated as a biological control agent for Avena species (wild oats). Conidium production was affected by nutrition, pH, temperature and light conditions. Of the agar media tested, Czapek Dox agar (CZA) and half-strength oatmeal agar (½OMA) were the only media where sporulation occurred at all temperatures tested under a 12-h light:12-h dark photoperiod (L/D). The optimum temperature for conidium production was 20°C on ½OMA, whereas there was no optimum temperature on CZA. Under a 12-h near-ultraviolet (NUV):12-h dark photoperiod (NUV/D), similar numbers of conidia were produced on CZA at 6.66, 14.56, and 22.78 W m^?2, whereas on ½OMA conidium production was the highest at 14.56 W m^?2. When NUV/D and L/D conditions were compared, similar numbers of conidia where produced on CZA, whereas ½OMA conidium production was superior under the NUV/D photoperiod. Considerable variation in sporulation and degree of virulence of D. avenacea was detected among isolates from different geographic areas. The most virulent conidia were obtained on ½OMA at 20°C incubated under continuous illumination NUV light. Therefore, the most suitable conditions for conidium production of D. avenacea were growth for 1 week on ½OMA at 20°C under continuous NUV at an intensity of 14.56 W m^?2. Under these conditions, 1.1×10⁵ conidia mL^?1 were produced which is the highest sporulation yet reported for any Drechslera spp., which are traditionally poor sporulators.     

Development of a submerged-liquid sporulation medium for the johnsongrass bioherbicide<Emphasis Type="Italic"> Gloeocercospora sorghi</Emphasis>

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Gloeocercospora sorghi. In phase I, 18 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, butter bean and lima bean brines (1.5–4.6 mS/cm) provided best conidiation. In phase II, a fractional-factorial design was utilized to screen 76 different medium adjuncts in combination with butter bean brine for improved sporulation. d-Mannitol and carboxymethylcellulose (CMC) were the only acceptable factors that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 2.69 mS/cm butter bean brine +0.043 M d-mannitol +0.37% w/v CMC with an expected titer of 1.51×10⁷ conidia/ml. Actual mean titer attained with the model-derived medium was 1.91×10⁷ conidia/ml. Optimal sporulation occurred at 25.5°C in this medium and conidia remained viable up to 2.71 days when stored at 12°C. No significant difference was observed in virulence of conidia produced on agar vs washed conidia produced in the model-derived (liquid) medium.     

Development of a submerged-liquid sporulation medium for the potential smartweed bioherbicide Septoria polygonorum

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Septoria polygonorum. In phase I, 47 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, pea brine (5–10% v/v) provided best conidiation. In phase II, a fractional factorial design was utilized to screen 38 different medium adjuncts in combination with pea brine for improved sporulation. MgSO₄ was the only factor that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 8.88% v/v pea brine+0.1 molar MgSO₄ with an expected titer of 1.78×10⁸ conidia/ml. Actual mean titer attained with the model-derived medium was 1.15×10⁸ conidia/ml. No significant difference was observed in virulence of conidia produced on agar vs. the model-derived (liquid) medium.     

Distribution,occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area

The occurrence of entomopathogenic fungi was investigated in irrigated vegetable fields and citrus orchards soils, over a nine-month period (April-December 1999),using the Galleria bait method (GBM). Entomopathogenic fungi were found to occur in 33.6% of the soil samples studied, with positive samples yielding 70 fungal isolates, belonging to 20 species from 13 genera. Conidiobolus coronatus was the most frequent and abundant entomopathogenic species recovered, comprising 31.4% of the total number of isolates. Soil pH, soil moisture content and the geographical location had minor or no effect on the isolation of entomopathogenic fungi in the fields studied. On the other hand, organic matter content of soil, and vegetation type were found to significantly affect the occurrence of entomopathogenic fungi in soil habitats, with orchard fields yielding larger numbers of isolates than vegetable fields. Using Koch's postulates the pathogenicity of fungal isolates to Galleria larvae was found to range from 16–100% (mortality rate). Isolates of C. coronatus proved to be the most virulent isolates recovered. The effect of media and temperature on mycelial growth rate, conidial production and conidial germination of six entomopathogenic fungal species (C. coronatus, Entomophaga grylli, Erynia castrans, Hirsutella jonesii, Paecilomyces farinosus and Sporodiniella umbellata) was also studied. Mycelial growth rate, spore production and spore germination were significantly affected by media, temperature and isolates. In view of the present results, C. coronatus appears to be a good candidate for pest control in agricultural soils, as it has a wide tolerance to agricultural practices, has frequently been isolated from both vegetable and orchard fields, and is characterized by high mycelial growth rate, conidial production and conidial germination.This revised version was published online in October 2005 with corrections to the Cover Date.     

A new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, cause of foliar spot blotch in wheat and barley

We developed a new technique for monoconidial culture of the most aggressive isolate in a given population of Bipolaris sorokiniana, to facilitate the evaluation of spot blotch resistance in wheat and barley. Blotched portions of infected barley leaves were placed on a glass slide in a moist chamber for production of conidia by associated fungal hyphae. Conidia were collected separately and grown on water agar discs. Individual water agar discs having conidium growth were inoculated on barley leaves. The conidium producing the earliest symptom with the largest lesion was considered most aggressive. This lesion was incubated in a moist chamber and the conidial offspring were tested for pathogenicity. When a uniform infection was observed, a small piece of the lesion was cut using a sterilized scalpel, surface sterilized with NaOCl, and inoculated in the centre of Petri dishes containing potato dextrose agar medium. The inoculated Petri dishes were incubated at 25 ± 1 °C to yield monoconidial cultures of the most aggressive isolate. Variability in symptom expression caused by the most aggressive isolate of a given population was much less than variability in symptom expression caused by all isolates collectively. The techniques will be useful for plant pathologists and breeders in screening for spot blotch resistance in wheat and barley.     

Optimising inoculum yield and shelf life of Plectosphaerella cucumerina,a potential bioherbicide for Cirsium arvense

Plectosphaerella cucumerina was identified as a potential bioherbicide for controlling Cirsium arvense in Canada and New Zealand. The current study evaluated production conditions using two isolates (one from each country) to determine whether the yield and shelf life of inoculum are suitable for mass production. Mycelial growth and sporulation in culture both increased from 15°C to 25°C and declined at higher temperatures with no mycelial growth at 37°C. The Canadian isolate produced fewer conidia than a New Zealand isolate. Potato dextrose-based liquid media with moderate to high concentrations of carbohydrates (25%, 50%, and 100%) maximised conidia production and these base media produced conidia with the highest germination rate (>80%) both at harvest and after 4 weeks stored at 4°C in 2.5% glycerol, 40% milk glycerol or after air drying. However, after 10-week storage, the conidia failed to germinate. Sporulation occurred during growth on all solid substrates tested (rice, rolled barley, and triticale), but conidial germination was highest on rice and barley, both before and after air drying. By contrast to conidia, 90% of mycelia-infested barley grains were viable after 3 years of storage at room temperature, although viability was lost by this time on the other substrates. This study has shown that the nutritional base is an important determinant of sporulation and shelf life for P. cucumerina. Although the yield of conidia in liquid medium was adequate to justify further development of P. cucumerina as a bioherbicide, improvement in its shelf life, or alternate formulation types that extend the shelf life, must be made for commercial efficiency.