| Development of plant pathogenic fungi as bioherbicide, which was isolated from alien invasive weed, will be one of the most effective and eco-friendly technology for weed management. The Sclerotium rolfsii strain SC64, firstly isolated from an invasive alien weed Solidago canadensis, was virulently pathogenic to this weed and a lot of other broadleaf weeds. This research includes:1. Isolation and identification of the pathogenic fungi from diseased Canadian goldenrod and four other plants. Five strains SC64, CSC, DSC, HSC and QSC, identified as Sclerotium rolfsii based on morphological characters, was isolated from damaged plants with a destructive stem rot disease symptom sporadically occurring on Canadian goldenrod (Solidago canadensis L.), Bryophyta; Chlorophytum comosum; Arachis hypogaea and Begonia evansiana in Nanjing and Jiangdu city, Jiangsu province of China. ITS sequence (684,687,681,685 and 665 bp) analysis through initial amplifying from the pathogen with the universal primers ITS1 and ITS4, and comparing with those of related species acquired from GenBank database confirmed the identification of these strains. Sclerotia serve as the principle overwintering structures and primary inoculum for disease. Persisting near the soil surface, sclerotia may exist free in the soil or in association with plant debris. S. rolfsii, the first identified causal agent of stem rot on Canadian goldenrod in China, may have potential to be developed into a bioherbicide for this weed and other broadleaf weeds.2. The host range of isolate SC64. A total of 105 plant species in 29 families were screened against isolate SC64 in host range studies. Most tested species of broad-leaved weeds and crops with economical importance in Dicotyledonae and four (Allium sativam L., Cyperus difformis L., Commelina bengalensis L. and Commelina communis L.) in Monocotyledonae were susceptible to strain SC64, but no mortality or significant disease symptoms were observed for turf grasses and grain crops. The most susceptible families dominated in Asteraceae, Scrophulariaceae, Convolvulaceae and Leguminosae, but all tested species were tolerant in the family of Chenopodiaceae, Phytolaccaceae and Poaceae. In some families, only partial species was susceptible to isolate SC64, such as Amaranthaceae, Polygonaceae, Apiaceae, Portulacaceae.3. The infection process of mycelium of isolate SC64. (1) Histopathology of infection of Canadian goldenrod stems by S. rolfsii strain SC64 was studied by using paraffin section observation and scanning electron microscopy. About 12 h after inoculating stems with mycelium suspension, dense mycelial net works were usually formed on the inoculated tissues. Then appressoria were developed by expansion at the tip of a single hypha, which wored as infection cushions. Hyphae forming infection cushions were often flattened and increased in diameter. Mucilage produced by the pathogen covered hyphae and infection cushions. Infection hyphae developed ramifying hyphae that differed in size and appearance from infection hyphae and then withered and collapsed. Penetration occurred after death and collapse of cells around infection cushions and from ramifying hyphae that developed from individual infection hyphae. Overall, the infection process included formation of infection cushions on the surface by expansion at end of single hypha, mechanical penetration from the host epidermis, development of infection hyphae intercellularly in the epidemic cells, inter-and intracellular development of ramifying hyphae. Infection cushions played a pivotal role during penetration and mechanical pressure also played an important auxiliary function.(2) Histopathology of infection of Cyperus difformis and six other Cyperus species stems by S1. rolfsii strain SC64 were studied by using light and scanning electron microscopy. Hyphae from mycelium suspension of strain SC64 ramified over various host tissues within 12 h following inoculation. Only C. difformis was susceptible to strain SC64. Results revealed that host tissues were infected by S. rolfsii SC64 by direct hyphal penetration through epidermis or wavelike hyphae crossed the spines and reached the place of stromata and broke into interior tissues. Mucilage produced by the pathogen covered hyphae and infection sites. Differences of the main micro-morphology characters of leaf abaxial epidermis and transverse sections of the 7 Cyperus species were compared. The stomata of C. difformis were always present between the leaf veins (3 or 4 rows of cells from the leaf veins) while the stomata of resistant Cyperus species were close to the leaf veins. Underneath the stromata of C. difformis were air space and vascular bundles were always present underneath the stomata of non-susceptible Cyperus. The differences of C. difformis were beneficial for infection by S. rolfsii SC64. The study suggests that fungal secretion (oxalic acid and cell wall degradation enzymes) and the special stomata location and structure of C. dofformis may play a role in the infection process.4. The biological characteristics of isolate SC64. Factors influencing mycelial growth and sclerotial germination of strain SC64 of S. rolfsii were studied. The results indicated that the most optimum media for the mycelial growth were modified Richard and potato dextrose agar (PDA), the range of temperature for the mycelium growth was 15～40℃ the optimum at 30℃, the range of pH was 4.0-6.0, with the optimal pH of 5.0. Through carbon sources testing on amended Czapek, sucrose was found to support growth significantly faster than other sources, while lactose was found to provide the slowest growth. Among the 13 nitrogen sources tested, peptone and potassium nitrate gave the best growth whereas urea gave the least growth. The range of temperature for sclerotia germination was 15～40 ℃, the optimum at 30℃. Sclerotia germination reached 100% when incubated on bran substrate with≥50% moisture. The range of pH for sclerotia germination was 2-10, with 100% germination at 3-9. Hyphal and sclerotia viability was completely lost when hyphal agar plugs and sclerotia incubated at 45 ℃ 10 minutes and 50℃ 10 minutes, respectively.5. Factors affecting liquid fermentation of isolate SC64. The optimal conditions for liquid starter culture fermentation and the effects of environmental factors and three precursors (citric acid, ascorbic acid and sodium succinate) on mycelial growth, oxalic acid production and virulence were studied. The results showed that for mycelia growth the optimum liquid medium was Modified Richard’s solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached S. canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg·g-1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27 ℃, but increased mycelial dry weight and virulence were observed at 30℃. The optimum range of initial pH value for oxalic acid accumulation was 4.0-6.0, with the optimal pH of 5.0; highest mycelial growth was with an initial pH 3.5-6.0, (optimum pH of 5.0) and subsequently pH 3.5-5.5, (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high should be useful for further development of this fungus as biocontrol agent.6. Cultivation and application of S. rolfsii strain SC64 for the mass-production of fungus-infested substrates by using agricultural wastes. The effects of different solid substrates, combination of substrates on efficacy of fermentation, storage, activation of mycelium and bioherbicidal assay under greenhouse conditions of fungus-infested substrates were investigated. Results showed that, among the 12 solid substrates, bran and cotton seed hulls supported maximum mycelium growth, with oxalic acid accumulation and lesion area on detached croftenweed leaves 6.06,5.94 mg/g and 2.78,2.73 cm2, respectively. When adding organic fertilizer (cottonseed mashroom cultural waste) to wheat-ear substrates (of 40% w) or bran to rice husk (of 50% w), oxalic acid accumulation and lesion area on detached croftenweed leaves were equal or overcome that of fungus infested bran. Optimal water/substrate ratio for organic fertilizer-wheat-ear and bran-rice husk compound substrates was 100～110% and 30%, respectively. When using fungus-infested cottonseed hulls as application material, optimal conditions for mycelium germination and growth on soil surface were 25% soil moisture content, pH 5and 25 ℃; together inner soil were 20% soil moisture content, pH 7and 30 ℃; fresh sclerotia germination were 15% soil moisture content, pH<6 and 25℃. Preliminary bioassays showed that isolate SC64 caused 70.5-87.7% mortality rate and 78.7-91.6% fresh weight reduction in five serious weeds inoculated with fresh fungus-infested rice husk-bran(2:1, v/v) at the rate of 140 (or 120) g·m-2 under greenhouse conditions; isolate SC64 caused 41.6%,45.2% and 84.4%,76.3% of mortality rate and fresh weight reduction of Amaranthus retroflexus and Digitaria sanguinalis when treated pre-emergence,14.3%, 25.9% and 47.0%,58.5% of mortality rate and fresh weight reduction when treated at 7 or 2 true leaves stage, respectively.7. The effect of environment factors on the pathogenicity of isolate SC64. The effect of host-plant (eclipta (Eclipta prostrata) as a model) growth stage, dosage of inoculum, soil moisture condition, temperature, and damp soil duration was studied in pot experiments. The results showed that before 5 leaf stage,100 g·m-2 fungus-infested substrates (rice husk/bran substrate,2:1, v/v),27～33 ℃ of temperature, soil of 90% water holding capacity and at least 24 h damp soil duration were essential for perfect control effect. A simulation experiment of dry-seeded rice was carried out to examine the performance of S. rolfsii SC64 to broadleaf weeds under greenhouse conditions. Results showed that isolate SC64 (120 g·m-2) caused 80% and 75% plant mortality,84% and 83% fresh weight reduction for Rotala indica and Monochoria vaginalis, respectively. Plant mortality and fresh weight reduction for Cyperus difformis and Ammannia arenarid were relatively lower, which were approximately 50% and 60-65%, respectively. Therefore, this fungus appeared to be a good candidate for further studies and a promising biocontrol agent to broadleaf weeds.8. Evaluation of the control efficacy of isolate SC64 in the field trials. (1) The fungal pathogen Sclrotium rolfsii isolate SC64 is being assessed as the basis for a mycoherbicide for biological control of broadleaf weeds in dry-seeded rice. Species tested for susceptibility in the field included Cyperus difformis, Lindernia procumbens, Rotala rotundifolia, Ammannia baccifera and Eclipta prostrata. Following preliminary small plot field applications in summer 2008/10, applications of fungus-infested solid substrates (mixture of rice hulls and bran) of 60 to 140 g·m-2 were conducted at two sites in Jiangsu province of China in summer 2010. The sites included a 1 year rested field and a wheat-rice rotation field. Plant mortality was recorded 7 and 14 days after inoculation (DAI). Percentage mortality ranged from 50 to 89% and 30 to 71% in the 2008 and 2010 solarium small plot trials, respectively. At the Nantong site field trial,30 to 60% plant mortality and 31 to 59% fresh weight reduction were recorded at 14 DAI when applied for the first time but the efficacy increased to 39 to 86% and 42 to 90% for plant mortality and fresh weight reduction at 14 DAI with a repeated application. Higher levels of plant mortality (42 to 77%) and fresh weight reduction (52 to 82%) were achieved at 14 DAI at the Rugao site with once treatment, due to the lower weed density and more favourable temperature and humidity conditions at the time of pathogen application. Simulation experiments in greenhouse have revealed these sclerotia do not survive over 60 days because of the relatively high temperature (average 30℃) soaked environment. Results confirmed that S. rolfsii SC64 is a potential biocontrol agent of some of the broadleaf weeds tested in dry-seeded rice.(2) To develop a rapid non-chemical control strategy for this weed in heterogeneous environments, this report explored different methods include cutting, plowing and treating with an indigenous pathogen S. rolfsii SC64, which was isolated from S. canadensis and applied by using solid formulation. We conducted field trials to test how controlling regime (i.e. initial treatment method, combination of these methods and treatment timing) influence the control efficiency. Results showed that simply cutting, plowing or treating with S. rolfsii SC64 does not sufficient to control S. canadensis. Cutting treatment performed in July and September eliminated sexual reproduction of S. canadensis. Combination of cutting, plowing and treating with isolate SC64 during growing season in May, July and September could kill more than 90% ramets. This treatment not only eliminated sexual reproduction of S. canadensis, but also damaged its underground stems. Therefore, this treatment may develop to be an optimized control strategy for S. canadensis.(3) Experiments on field weed control efficacy of S. rolfsii strain SC64 against broadleaf weeds on lawn were studied. The results showed that, with inoculum of 100 g·m-2 at 14 DAI, strain SC64 could provide 75% weed mortality and 88% fresh weight reduction, respectively. With the same dosage used on three different lawns, Festuca arundinacea; Cynodon dactylon and Zoysia matrella, strain SC64 could reduce over 80% broadleaf weed density. There was no visual damage to lawn weeds. These results indicate that strain SC64 is a potential microbial bioherbicide for control of broadleaf weeds in lawn.(4) S. rolfsii is being evaluated as a possible bioherbicide for control of Digitaria sanguinalis and Amaranthus retroflexus in corn and Eclipta prostrata and other broadleaf weeds in dry, direct-seeded rice. Granular fungus-infested substrates were applied pre-emergence into the soil under both controlled and field conditions. Under a controlled environment (corn), a granular bran-rice husk formulation (60～140 g·m-2) of the fungal inoculum that had been applied directly into soil only resulted in 20～35% plant mortality and 22-35% above-ground biomass reduction (21 DAI). Higher levels of plant mortality (23～49%) and fresh weight reduction (22～52%) were achieved at 28 DAI when wheat straw amended to soil combined inoculation with fungus-infested substrate of S. rolfsii SC64. A dose response of wheat straw amended to soil was also examined. When amended with 1 x and 2x of regular wheat straw to soil, application of 100 g·m-2 S. rolfsii resulted in above-ground biomass reduction of up to 47%,63% and 47%,68% for D. sanguinalis and A. retroflexus, respectively. This was confirmed in a small-plot field trial, which resulted in above-ground biomass reduction of up to 45%,59% and 51%,64% for D. sanguinalis and A. retroflexus, respectively. Following preliminary pot applications, applications of fungus-infested solid substrates of 60～140 g·m-2 were conducted at Nantong in dry, direct-seeded rice with wheat straw amended into soil in summer 2011.51 to 78% plant mortality and 45 to 75% fresh weight reduction were recorded at 28 DAI. During these trials, there was no effect on corn (or rice) germination and growth. Findings in this study indicated that S. rolfsii may provide effective control of weeds when combined with wheat straw amended to soil.The strain SC64 was systematically evaluated as a potential bioherbicide by using fungus-infested substrates as application material. The research contents included host range, infection process, biological characteristics, liquid culture establishment, solid fermentation, infection influence factors, and field application of the strain. The experiment provided a new method to biocontrol S. canadensis as well as broadleaf weeds in gramineous crop fields, turfgrass and landscape habitats. |
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