Demographic and Biomass Production Consequences of Inundative Treatment of Cirsium arvense with Sclerotinia sclerotiorum

The adventitious shoots in three populations of Cirsium arvense in sheep-grazed pastures were treated in October (spring) 1991 with a mycelium/wheat formulation of Sclerotinia sclerotiorum and the fates of mapped shoots were followed over the growing season. In untreated plots, deaths through natural causes were compensated for by births (emergence of new shoots above the soil) throughout the growing season, but, on plots treated with S. sclerotiorum, deaths from the induced disease exceeded births for 35 days following treatment, causing the shoot population to decline markedly. Disease-induced deaths occurred only among shoots present at the time of treatment; there was no evidence of transfer of the pathogen to shoots emerging after the treatment was applied. A life-table analysis showed that only 8% of the adventitious shoots emerging during the growing season survived to seeding on treated plots, compared with 28% on the untreated plots; most mortalities occurred in shoots at the vegetative stage of development. The dry mass of propagative roots in autumn was reduced to 35% of that on the untreated plots by the pathogen and the density of shoots emerging the following spring was reduced to a similar extent. The results of this study indicate that S. sclerotiorum has potential as a mycoherbicide for C. arvense in sheep-grazed pasture in New Zealand.     

Favourable Conditions for the Bioherbicide Candidate Fusarium tumidum to Infect and Cause Severe Disease on Gorse (Ulex europaeus) in a Controlled Environment

The development of the pathogenic fungus Fusarium tumidum on gorse ( Ulex europaeus ), a major weed of pastures and plantation forests in New Zealand, was studied under controlled conditions. F. tumidum , like most other foliar fungal pathogens, requires moisture to infect plants. Long, continuous dew periods ( 24 h) after inoculation of plants provided favourable conditions for infection. The fungus, however, also caused severe disease on young plants (2 months old) exposed to two or three 12-h dew periods interrupted by 12-h dry periods. A delay of 24 h before inoculated plants were exposed to dew did not affect the severity of the disease. F. tumidum infected plants over a wide range of temperatures (5-27IC), but more plants were killed as temperatures increased during the initial infection phase. All gorse plants tested (up to 4 months old) were susceptible to the fungus, but younger plants were more easily killed. Nevertheless, the biomass of older plants that were severely diseased but not killed by the fungus was significantly reduced. The effectiveness of F. tumidum in killing plants increased with the density of inoculum sprayed. The fungus applied at a density of 1 106 conidia/ml killed more than 95% of 1.5-month-old plants. This basic knowledge of the F. tumidum -gorse system will assist in the development of a pilot bioherbicide to control gorse and broom ( Cytisus scoparius ), another economically important weed in New Zealand which is also susceptible to the fungus.     

Isolation, Pathogenicity and Safety of Curvularia eragrostidis Isolate QZ-2000 as a Bioherbicide Agent for Large Crabgrass (Digitaria sanguinalis)

A pathogen isolated from lesions on blighted leaves of crabgrass in three different locations of China was identified as Curvularia eragrostidis. Isolate QZ-2000 was the most virulent of six isolates tested. Experiments on morphology, pathogenicity, effect of environmental factors, and host-range of isolate QZ-2000 were conducted in the laboratory, greenhouse and field to assess the potential of this isolate as a biocontrol agent for grassy weeds. Pathogenicity was quantitatively determined based on mortality and dry-weight reduction of infected large crabgrass. Inoculum concentration, rapeseed oil concentration in formulaton, post-inoculation dew temperature and duration, and plant growth age all significantly influenced the efficacy of the isolate. A total of 85-100% control of large crabgrass was obtained when inoculum concentrations were ≥1×10⁶ conidia mL^-1, oil concentrations ≥0.9% (v/v), dew period ≥24 h and air temperatures 20-30°C in the greenhouse. A total of 51 plant species in 20 families were screened against isolate QZ-2000 in host-range studies. Six other species of grassy weeds were susceptible to isolate QZ-2000, but no mortality or significant dry-weight reduction was observed for maize (Zea mays), rice (Oryza sativa), soya bean (Glycine max), cotton (Gossypium hirsutum), or any other economically important crops and plants. In field trials, with 5×10⁶ conidia mL^-1 inoculum density, 60-7% reduction in dry weight was achieved for large crabgrass seedlings under natural dew-free conditions. These results indicate that isolate QZ-2000 is a potential microbial bioherbicide for control of large crabgrass in crops such as corn, soybean, cotton, water-melon, and peanut.     

Fusarium oxysporum f. sp. strigae strain Foxy 2 did not achieve biological control of Striga hermonthica parasitizing maize in Western Kenya

The production of maize, a major staple food crop in sub-Saharan Africa is being constrained by the parasitic weed Striga hermonthica. The fungus Fusarium oxysporum f. sp. strigae (Foxy 2) that causes fusarium wilt of Striga in Ghana, West Africa, is being considered for biological control of the weed in Western Kenya. The present study investigated the efficacy of F. oxysporum f. sp. strigae (Foxy 2) for S. hermonthica management in Western Kenya. Research was conducted in post-entry quarantine (PEQ) facilities at Alupe, Busia, Homabay, Kibos and Siaya field stations for two seasons. Each PEQ was a split-plot, with 4 main blocks each having 6 treatment subplots. The treatments included seeds of two S. hermonthica-susceptible maize varieties, either coated with Foxy 2 using gum Arabic, gum Arabic alone, or left untreated. Data was collected over seven sampling periods on S. hermonthica population per plant, percentage of those that were wilting, and the severity of wilting. Maize plant growth parameters assessed included duration to 50% anthesis and 50% silking, plant height, number of leaves, stover and cob weights, and maize yield per hectare. Statistical analysis was done using SAS 9.1 software. Data on S. hermonthica population were analyzed by χ²-test using Proc Genmod (Poisson); while the other parameters were analyzed by Proc Mixed using study location, season and blocks as random effects, and the sampling periods as repeated effects. All the assessed parameters were similar between plants grown from seeds inoculated with F. oxysporum f. sp. strigae (Foxy 2), those coated with gum Arabic, and the ones without any coating. These parameters were also not different between the maize varieties. There are varying reasons for the disparities between results on F. oxysporum f. sp. strigae (Foxy 2) obtained in this Kenyan study, and those from researches outside this country. In conclusion, F. oxysporum f. sp. strigae strain Foxy 2 is predominantly safe on maize growth, but its efficacy in controlling S. hermonthica was not evident on the tested Kenyan soils.     

Hydroponic Seedling Bioassay for the Bioherbicides Colletotrichum truncatum and Alternaria cassiae

A rapid bioassay was developed to measure the bioherbicidal efficacy of spore preparations of the pathogens Colletotrichum truncatum (Schwein.) Andrus and W. D. Moore and Alternaria cassiae Jurair and Khan on hemp sesbania (Sesbania exaltata) and sicklepod (Cassia obtusifolia), respectively. The system uses 4-day-old dark-grown seedlings (grown hydroponically in paper towel cylinders) which were sprayed with spore suspensions. Shoot lengths were monitored non-destructively, and recorded over time under conditions of dark growth, 90-100% relative humidity and 25 C. Shoot growth inhibition and stem collapse (mortality) were directly related to the spore concentration applied. Generally, at 10 3 - 10 4 spores ml-1, these pathogens caused significant shoot growth inhibition within 25-30 h and seedling death within 40-50 h. This bioassay has been used to study herbicide-pathogen interactions, and may be extended to determine the bioherbicidal efficacy of different pathogen isolates, pathovars or spore formulations. This technique is more rapid, uses a lower inoculum volume, requires less space and is performed under more controlled conditions than conventional greenhouse bioassay methods. The data obtained are more quantitative than those obtained from bioassays relying on visual rating systems.     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

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.     

Chlamydospore Production,Inoculation Methods and Pathogenicity of Fusarium oxysporum M12-4A,a Biocontrol for Striga hermonthica

Fusarium oxysporum isolate M12-4A is currently being evaluated for the biological control of Striga hermonthica . Inoculum production, inoculum delivery to the target, chlamydospore germination, and weed growth suppression of this weed-pathogen system were investigated. Liquid fermentation systems using organic material were evaluated for the production of large numbers of chlamydospores. A 1% sorghum straw powder (< 1 mm) substrate, exposed to black light at 21°C for 21 days, yielded 3.23 X 10 8 colony forming units (CFU) l -1 medium. A two-stage fermentation system using 5% w/v straw substrate under black light at 30°C for 14 days yielded 3.5 X 10 8 CFU l -1 medium. In vitro variations in chlamydospore germination were governed by the presence of exogenous carbon, nitrogen, and sorghum root exudates. Ammonium-nitrogen compounds and urea, in combination with glucose had a stronger stimulatory effect on chlamydospore germ tube growth than did potassium nitrate. Maximal germ tube elongation occurred when chlamydospores were exposed to urea at a C/N ratio of 10. Some mineral solutions and sorghum root exudates inhibited chlamydospore germ tube elongation; however, arabic gum, a complex polysaccharide, stimulated chlamydospore germ tube elongation and the production of secondary chlamydospores. In field trials, chlamydospore powder harvested from small-scale fermenters reduced S. hermonthica emergence by 92%. Complete inhibition of S. hermonthica emergence occurred when the chlamydospore powder was added to the soil at sowing and when sorghum seeds coated with chlamydospores were sown. Effective biological control of S. hermonthica was achieved using a simple fermentation system with sorghum straw as the inoculum growth substrate. For inoculum delivery to the farmers' fields, sorghum seeds were coated with the inoculum using arabic gum as the adhesive. This simple delivery system permits a uniform inoculation of the field as well as the proper positioning of the inoculum in the immediate environment of sorghum roots, where S. hermonthica attaches to its host. To facilitate a broad usage of F. oxysporum M12-4A for the biocontrol of S. hermonthica , we propose an inoculum production strategy based on a cottage industry model that utilizes a liquid fermentation process and inexpensive locally-available substrates including sorghum straw and arabic gum.     

Conidial germination and germ tube elongation of Phomopsis amaranthicola and Microsphaeropsis amaranthi on leaf surfaces of seven Amaranthus species: Implications for biological control

Microsphaeropsis amaranthi and Phomopsis amaranthicola are potential biological control agents for several Amaranthus species. In an effort to understand the initial infection processes with these pathogens, a study was conducted of the conidial germination and germ tube length (μm) on the weed leaf surfaces at 21 °C and 28 °C. Weeds included Amaranthus rudis, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. hybridus, and A. albus. For P. amaranthicola, conidial germination and germ tube length varied among the seven weed species at both temperatures, while for M. amaranthi the differences in germ tube lengths were significant among weed species only at 21 °C. While the conidia of M. amaranthi and P. amaranthicola germinated on the leaf surfaces of all seven weed species, temperature appeared to impact the number and length of germ tubes on the leaf surfaces. The percentage of germinated conidia and the length of germ tubes at both temperatures were often greater for M. amaranthi than for P. amaranthicola. In order for the fungal pathogen to successfully infect and kill a weedy host, conidia must germinate and form a germ tube, two processes that vary with host species and temperature for M. amaranthi and P. amaranthicola. The extent to which successive infection processes, e.g., penetration, invasion and colonization, contribute to host specificity warrants study.     

Water Activity and Other Factors that Affect the Viability of Colletotrichum truncatum Conidia in Wheat Flour-Kaolin Granules ('Pesta')

Optimization of shelf-life is critically important for biocontrol products containing living microorganisms. Conidia of Colletotrichum truncatum, a fungal pathogen of the weed, hemp sesbania (Sesbania exaltata), were produced in shake flasks (corn meal-soya flour medium) and on Emerson Yp Ss agar and formulated in wheat flour-kaolin granules ('Pesta'). The granules were conditioned at water activities of 0, 0.12, 0.33, 0.53 and 0.75 during storage at 25 C over desiccant or saturated salt solutions. The longest shelf-life (conidial inoculum viability) was found in samples in the water activity range 0-0.33, where the water was bound by the matrix and not readily available to the fungus. At a water activity of 0.12, granules were 100% viable (on water agar) for at least 24 weeks, and were 87% viable after 1 year. Sucrose (5% w/w) partially counteracted the detrimental effect of high water activity on the shelf-life of C. truncatum when incorporated in the granules.