Abstract:
The invention disclosed relates to a naturally occurring fungus,  Valdensinia heterodoxa , and to its culture, formulation and delivery systems, as well as its use as a biocontrol agent for salal ( Gaultheria shallon  Pursh.).

Description:
BACKGROUND OF THE INVENTION  
         [0001]    The present invention relates to the naturally occurring fungus,  Valdensinia heterodoxa , and in particular to its use as a biocontrol agent for salal ( Gaultheria shallon  Pursh.).  
           [0002]    Forest management has become increasingly intensive in order to maximize forest productivity and sustainability. The past few decades have seen significant changes in forest management practices, especially in the area of site preparation, the use of chemical herbicides, and development of new forest harvesting systems. Competition from non-commercial or competing forest vegetation is a major problem at conifer regeneration sites following harvest in plantations. This competition results in conifer mortality, reduced growth, delays in harvesting time, increased costs related to forest management, and decreases in annual allowable cut (Wall et al. 1992). Management of competing forest vegetation can take various forms, including removal by mechanical or manual brushing and chemical herbicides. These methods have distinct disadvantages such as non-target effects and public concerns about the negative impacts of using herbicides in pristine forest ecosystems. Hence, there is a growing need for alternative management strategies for competing vegetation that are cost-effective, environmentally safe, economically feasible, and sustainable (Watson and Wall 1995). One viable option is the use of naturally occurring plant pathogens as biological control agents which, if successful, are expected to result in increased early conifer growth rate and a shorter rotation age of commercially valuable crop trees (Shamoun 2000).  
           [0003]    Salal ( Gaultheria shallon  Pursh.), a perennial, ericaceous shrub, is a serious competitor with conifer seedlings in coastal British Columbia. Generally, it competes with trees for water and nutrients and removal of salal leads to enhanced conifer growth (McDonald 1990). Salal is difficult to control with current mechanical methods due to its extensive root system leading to quick reestablishment through layering, sprouting, and suckering (D&#39;Anjou 1990). Chemical herbicides are often ineffective since salal&#39;s thick and leathery leaves reduce herbicide translocation (D&#39;Anjou 1990). Hence, salal can be considered a suitable target weed for biological control using fungal pathogens.  
           [0004]    Numerous fungal species have been isolated from salal plants, including  Mycosphaerella gaultheriae  (Haeussler et al. 1990),  Phyllosticta pyrolae  Ellis et Everh. (Petrini et al. 1982),  Phytophthora cinnamoni  Rands (Lindermann &amp; Zeitoun 1977), and  Valdensinia heterodoxa  Peyr. (Readhead 1974).  
           [0005]    In 1999, a survey was conducted to collect and identify the mycobiota associated with salal from various locations on Vancouver Island (Shamoun et al. 2000).  
           [0006]    Fungal pathogens isolated from diseased leaf and stem tissue were subsequently assessed for their virulence on salal. From the tested fungi,  Valdensia heterodoxa  (PFC 3027) caused substantial leaf damage on both detached leaves and intact salal plants (Vogelgsang et al. 2001).  
         SUMMARY OF THE INVENTION  
         [0007]    According to an embodiment of the present invention, a biologically pure isolate of the naturally occurring fungus,  Valdensinia heterodoxa , having all of the identifying characteristics of IDAC Deposit Accession no. IDA 180402, is provided.  
           [0008]    According to a further embodiment of the invention, a herbicidal composition containing as active ingredient,  Valdensinia heterodoxa , having all of the identifying characteristics of IDAC Deposit Accession no. IDA 180402, is provided.  
           [0009]    According to yet another embodiment of the present invention, a herbicidal composition for controlling salal ( Gaultheria shallon  Pursh) is also provided, the composition comprising as active ingredient, an effective amount of a culture of  V. heterodoxa  on an agriculturally and environmentally acceptable solid growth substrate capable of supporting growth of the fungus, containing a cereal grain, e.g., oatmeal.  
           [0010]    According to yet another embodiment of the present invention, a method for controlling salal ( Gaultheria shallon  Pursh.), is also provided, the method comprising applying to a salal plant or to a salal plant locus, an effective amount of a herbicidal composition containing as active ingredient, a biologically pure isolate of the fungus  Valdensinia heterodoxa  having all of the identifying characteristics of IDAC Deposit Accession no. IDA 180402.  
           [0011]    According to another embodiment of the present invention, a method is provided for isolating  Valdensinia heterodoxa  from nature in biologically pure form.  
           [0012]    According to yet another aspect of the invention, salal plant tisue e.g. leaves and stems colonized by  V. heterodoxa  is used as an inoculum delivery mechanism to control salal.  
           [0013]    According to yet another embodiment of the present invention, an inoculation of salal plant tissue, e.g., leaf pieces, with mycelium of  V. heterodoxa  produced from cereal grain, e.g., oatmeal, containing growth media e.g. agar.  
           [0014]    We have also found that using additional solid substrates infected with the fungus such as: 1) alder saw dust; 2) fir saw-dust; 3) vermiculite powder; 4) rice grain; 5) rolled oats; and 6) whole oats. Again, the results showed that the most effective solid substrate for formulation and conidia production and discharge was on salal leaves and stems. There was also production and discharge of conidia on alder saw dust and fir-saw dust, but was not significant compared to salal leaves and stems.  
           [0015]    Further, we have found that a medium containing rolled oats as the only carbohydrate source (e.g., water agar containing a few rolled oats on the surface) has triggered the sporulation of  Valdensinia heterodoxa.    
           [0016]    According to yet another aspect of our invention, we have found to advantage that innoculation of plant materials, such as leaves and stems as a delivery mechanism. Typically, in the prior art, non-host materials are used such as cereal grains, alginate pellets and vermiculite.  
           [0017]    As already mentioned above, in the culturing method itself, the use of oatmeal in the medium, lower incubation temperature and the use of host-tissue for final formulation and delivery technologies are novel for this pathosystem ( Valdensinia heterodoxa -Salal).  
           [0018]    It is also significant that there is a cut-off temperature above which sporulation is inhibited. Also, in the case of  Valdensinia heterodoxa , for conidia discharge, the optimum for conidia discharge at rather low temperature is unusual for the majority of fungal pathogens. More importantly, as will be apparent from the examples which follow, sporulation and discharge are severely inhibited above and below the determined optima.  
           [0019]    It is also interesting that alternating light and dark treatments resulted in significantly lower growth rates, sporulation, and conidia discharge. See Table 2.  
           [0020]    Deposit Information  
           [0021]    The above referenced Deposit was made with the International Depository Authority of Canada (IDAC), 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2, Canada, under the auspices of the Budapest Treaty. The Deposit was received by the IDAC on Apr. 18, 2002, and was tested and confirmed to be viable on Apr. 22, 2002.  
         BRIEF DESCRIPTION OF THE DRAWING  
         [0022]    The figure illustrates the effect of  Valdensinia heterodoxa  from colonized salal leaf pieces on intact salal plants 14 days post-inoculation (dpi). For each treatment, 3 g of uninoculated (control or colonized (PFC isolate 3027) leaf pieces were placed beneath salal leaves. First disease symptoms were observed at 4 dpi. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0023]    Isolation of  Valdensinia heterodoxa  from Diseased Leaf Samples  
         [0024]    [0024] Valdensinia heterodoxa  (PFC 3027) having all of the identifying characteristics of IDAC Deposit accession no. IDA 180402 was isolated from salal leaf tissues in biologically pure form, by surface sterilizing small pieces of infected tissue, plating them onto potato dextrose agar (PDA) or malt extract agar (MEA) and incubating in the dark at 20° C. Emerging colonies were subcultured onto PDA plates, and then transferred to PDA slants for long-term storage at 5° C.  
         [0025]    Starter Cultures of  Valdensinia heterodoxa    
         [0026]    Cultures of  V. heterodoxa  (PFC 3027) were initiated by placing small ( V. heterodoxa ) mycelium pieces from potato dextrose agar (PDA) slants maintained at 4° C. on salal PDA (SPDA; PDA amended with 40 g fresh, blended salal leaf and stem material/L dH 2 O). Fungal cultures were grown at 19/13° C. (day/night) with a 12 h day photoperiod (180-200 μEm −2 s −1 ). Eight days after inoculation, mycelial plugs of 5-mm diameter were transferred onto weak oatmeal agar (WOA; 15 g oatmeal agar and 12 g agar/L dH 2 O) and incubated at the desired conditions, depending on the experiment.  
         [0027]    Effect of Temperature on Growth, Sporulation, and Conidia Discharge of  Valdensinia heterodoxa    
         [0028]    Materials and Methods  
         [0029]    Growth chambers were programmed at 11/6, 14/9, 17/12, and 20/15° C., respectively, with a 12 h day −1  photoperiod (180-200 μEm −2  s −1 ). Fungal cultures of  V. heterodoxa  were initiated on SPDA and transferred onto WOA as described above. At 3, 6, and 9 days post-inoculation (dpi), mycelial radial growth (colony diameter), total number of conidia, and number of discharged conidia adhering to the lid of the Petri dish were determined. For each temperature treatment, five replicates were used and the experiment was performed twice. Data were subjected to a one-way analysis of variance (ANOVA). In case of failed normality or equal variance tests, a Kruskal-Wallis one-way ANOVA on ranks was used instead. Differences between treatment means were evaluated by a Student-Newman-Keuls multiple comparison procedure (P=0.05).  
         [0030]    Results  
         [0031]    Throughout the experiment, mycelial radial growth was greatest at the higher temperature regimes of 17/12 and 20/15° C. and was strongly inhibited at 11/6° C. Similarly, total sporulation was improved at higher temperatures. However, conidia discharge displayed a clear optimum at 17/12° C. and declined substantially at both temperatures above and below this regime. Differences in sporulation were observed as early as 3 dpi but more pronounced towards later evaluation dates. The results of both trials are summarized in Table 1.  
                                                                                                     TABLE 1                           Effect of day/night temperature on mycelial growth, sporulation, and conidia       discharge of  Valdensinia heterodoxa  (PFC 3027) at different days       post-inoculation (dpi)                Temperature   Trial 1   Trail 2                (° C.) 1     3 dpi 2     6 dpi   9 dpi   3 dpi   6 dpi   9 dpi                        Colony diameter   11/6    1.2 a    1.6 a    2.1 a    1.1 a    1.9 a    2.5 a 3         (cm)   14/9    1.7 b    2.9 b    4.4 b    1.5 b    2.9 b    4.1 b           17/12    2.0 b    3.5 b    4.6 b    1.9 c    3.6 c    4.7 c           20/15    2.0 b    3.5 b    4.7 b    1.6 b    2.1 a    2.4 a       Total number of   11/6    2.4 a    15.0 a    18.0 a   11.6 b    29.4 a    44.0 a       conidia   14/9   10.8 ab    23.2 a   108.6 a   12.4 b    48.8 a   174.0 a           17/12   23.6 b   121.2 b   318.0 b    3.0 a    206.4 b   862.4 b           20/15   26.6 b   158.6 b   381.6 b    0.0 a    4.2 a    13.2 a       Number of   11/6    0.0 a    0.8 a    0.8 a    0.2 a 3      1.6 ab 3      2.4 b 3         discharged   14/9    0.4 a    3.2 a    3.8 ab    0.6 a    3.4 ab    9.0 b       conidia   17/12    4.2 a    4.8 a    11.6 b    0.0 a    6.8 b    72.0 c           20/15    2.8 a    3.4 a    5.2 ab    0.0 a    0.0 a    0.2 a                                          
 
         [0032]    Effect of Photoperiod on Growth, Sporulation, and Conidia Discharge of  Valdensinia heterodoxa    
         [0033]    Materials and Methods  
         [0034]    Three photoperiod conditions including continuous light, alternating light/dark (12 h day −1  photoperiod), and continuous darkness were investigated. Cultures of  V. heterodoxa  were initiated on SPDA and transferred onto WOA as described above. Fungal cultures were grown in a single chamber programmed at 17/12° C. with 24 h day −1  light (180 μEm −2 s −1 ). The treatment of continuous darkness was achieved by wrapping the Petri dishes in aluminium foil. Petri dishes assigned to alternating light conditions were unwrapped daily at the start of the higher temperature cycle. Evaluation of mycelial growth, sporulation, and conidia discharge was performed as described above. For each photoperiod treatment, five replicates were used and the experiment was performed twice. Data were analyzed as for the previous experiment.  
         [0035]    Results  
         [0036]    Radial mycelial growth was moderately slower and total sporulation and conidia discharge were strongly inhibited by continuous darkness. Although all evaluated parameters were improved by a continuous light treatment, differences between and continuous and alternating light were not always significant (Table 2). As in the temperature experiment, differences were observed as early as 3 dpi. Sporulation data at later dates were highly variable for any of the light treatments. Results of both trials are summarized in Table 2.  
                                                                                                 TABLE 2                            Effect of photoperiod duration on mycelial growth, sporulation, and conidia       discharge of  Valdensinia heterodoxa  (PFC 3027) at different days       post-inoculation (dpi).                Trial 1   Trial 2 3                  Light treatment 1     3 dpi 2     6 dpi   9 dpi   3 dpi   6 dpi   9 dpi                        Colony diameter   Light    2.3 a    4.2 a    5.2 a    1.7 a    3.5 a    4.5 a       (cm)   Alternating    2.0 b    3.6 b    4.9 b    1.6 a    3.1 ab    4.1 a           Dark    2.0 b    3.4 b    4.7 b    1.6 a    2.8 b    3.7 b       Total number of   Light   64.8 a   428.2 a   865.0 a   48.4 a   227.6 a   519.0 a       conidia   Alternating    8.8 b   137.8 b   645.2 a    6.2 b    49.4 b   168.0 b           Dark    0.2 b    2.4 b    51.6 b    0.4 c    5.2 c    45.8 c       Number of   Light    5.4 a 3      35.0 a   126.4 a    3.0 a    13.2 a    17.2 a       discharged   Alternating    0.6 b    5.2 b    51.0 ab    0.6 b    3.6 b    5.2 b       conidia   Dark    0.0 b    0.0 b    1.6 b    0.0 b    0.2 c    4.8 b                                          
 
         [0037]    Solid-Based Formulation and Delivery Technique of  Valdensinia heterodoxa    
         [0038]    Materials and Methods  
         [0039]    Starter cultures of  V. heterodoxa  on SPDA were initiated as described above. Erlenmeyer flasks containing 250 mL of liquid weak oatmeal medium (WOM; 20 g blended rolled oats/L dH 2 O) were inoculated with 10 mycelial plugs from the starter cultures. Flasks were placed on a shaker (125 rpm) at 19/13° C. with a 12 h day −1  photoperiod (180-200 μEm −2  s −1 ).  
         [0040]    After 7 days, resulting mycelium was harvested on double-layered cheesecloth and blended in a Waring blender for 20 sec at high speed. Five mL of wet mycelium was added to Erlenmeyer flasks containing autoclaved salal leaf pieces (7 g litter+5 mL dH 2 O). The fungal inoculum was incubated as described above and flasks were shaken daily. After 14 days, the colonized leaf material was air-dried for 2 days in a fume hood.  
         [0041]    Salal seedlings were grown outside in 8 cm pots containing peat-vermiculite-sand (1:1:1) and a low rate of slow release fertilizer. Plants of similar size were selected and transferred into a growth chamber at the original conditions.  
         [0042]    Fungal inoculum was applied below salal leaves. For potted salal plants, usually several leaves protrude farther than the pot rim, hence, a structure was built beyond the rim to ensure that all leaves could be reached by discharging conidia. Plastic sheets (coroplast) were cut into 14×14 cm pieces with a 6×6 cm hole in the centre. To provide a rougher surface for the leaf pieces to be placed onto, cheesecloth was cut into the same outside dimensions as the coroplast with a ¾ slit in the centre. Both coroplast and cheesecloth were carefully placed around the plant and fixed with a pin. For each plant, 3 g of dried leaf pieces were applied onto the cheesecloth and watered with dH 2 O from a spraying bottle. Uninoculated leaf pieces served as the control treatment. For each treatment, five replicate plants were used. Pots were then transferred to a dew chamber (100% relative humidity, 20±1° C.) for 24 h; and subsequently covered with clear plastic bags and the ends tucked under the base of the pots. After the dew treatment, pots were moved back to the original growth chamber. Plastic bags were removed daily for a few minutes to ensure proper aeration. Leaf damage was evaluated 14 dpi, based on the percentage of necrotic leaf area. Data were analyzed using a Kruskal-Wallis one-way analysis of variance on ranks followed by a Student-Newman-Keuls multiple comparison procedure in order to evaluate differences between treatment means (P=0.05).  
         [0043]    Results  
         [0044]    First disease symptoms on salal plants developed 4 dpi and necroses expanded quickly over the next days, whereas no symptoms were observed on the control plants (Figure). Older leaves with thicker cuticle became infected as well, but necroses did not expand as fast compared with young leaves. The origin of the necrosis was easily identified by the discharged conidium still attached to the leaf surface in the centre of the necrosis. Some leaves with their upper surface facing down, developed disease symptoms as well. As the stomata of salal are only found on the lower leaf surface, it is assumed that conidia of  V. heterodoxa  are able to penetrate their host directly. In trial 1 and trial 2, plants exposed to the fungal inoculum developed on average 33 and 39% leaf damage, respectively.  
         [0045]    The observed leaf damage in this study is rather low compared with a spray application of other potential bioherbicides in which plants are usually completely covered with fungal propagules. Although most necroses developed rapidly, many leaves were not infected until the end of the experiment. On those leaves, very few conidia were found, due to either insufficient sporulation and/or loss of discharged conidia.  
         [0046]    The findings of this research confirm the potential of  V. heterodoxa  to be used as a biological control agent for salal, and in particular, the use of colonized leaf pieces as an inoculum source and delivery technique.  
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