GRASS TREATMENT

Abstract: VA mycorrhiza have growth-retardant activity for Poa annua and may be used to control the growth of P. annua in high quality sport or amenity turf consisting mainly of Agrostis or Festuca species. Strains of VA mycorrhiza selected from genera Glomus, Acaulospora, Entrophosphora, Gigaspora, Scutellospora and Sclerocytis may be used. The VA mycorrhiza is applied to the soil as a formulated product in which the VA mycorrhizae constitute the principal component having biological activity on the growth of turf grass. Dose rates of inoculum having an MPN of from about 200 To 1000 or more propagules per gram are effective and may be formulated with a clay or other solid carrier. The amount of formulated product applied to the turf may be from about 5 g/m<2> to about 1 kg/m<2>. The infective propagules are selected from fungal spores, mycelium, hyphae, and fragments of mycorrhiza infected roots. These may be applied to sport or amenity turf, or other grassy area in need of P. annua control, or an area in which high quality turf-grass is to be laid or sown.

Description: Grass Treatment This invention relates to methods and compositions for improving the quality of turf grass. More particularly, the invention is concerned with the control, reduction, or elimination of undesirable grass components of turf, especially of high quality turf consisting mainly of Bentgrass such as Agrostis stolonifera or Festuca species. Poa annua or Annual Meadow grass (AMG) (also called Annual Bluegrass) is the most troublesome weed of bent grass putting greens in countries as diverse as the United Kingdom, the United States of America, and Australia. P. annua is generally considered to be undesirable in putting greens because it is susceptible to abiotic stress, particularly water availability, as well as succumbing to a number of fungal diseases. In addition, if a green is composed of patches of AMG and bent grass, the surface is not as uniform as many players would wish it to be. Some turf managers attempt to control P. annua. The most widely used control method is herbicide application, and a number of compounds have been tested against the species with varying success. However, application of pesticides can be costly, and they may present a problem of groundwater pollution if the green is well irrigated. In addition, there may be associated health risks to golfers or greens staff. Therefore, if AMG is to be controlled on putting greens, a more natural, environmental approach is called for. 2 The present inventor has previously reported that the abundance of AMG in a golf green may be negatively related to the abundance of fungi in the soil (Gange A.C. 1994, Subterranean insects and fungi: hidden costs and benefits to the greenkeeper. In Science & Golf II, Proceedings of the World Scientific Congress of Golf, eds. A.J.Cochran and M.R.Farrally, 461-466, London E. and F.N. Spoon ). The fungi concerned were vesicular-arbuscular mycorrhizas which are generally abundant in natural plant communities. It was found that the fungi were very low in abundance in golf turf, but when they were common in the soil, there was less AMG in the sward, and vice versa. The original explanation for this relation was that as bentgrass is considerably more strongly mycorrhizal than AMG, then in greens where fungal abundance is high, the bentgrass is more vigorous, and therefore is able to out-compete the AMG. It has now been found that VA mycorrhizae have a direct growth- controlling effect on Poa annua and therefore provide an effective means of control of P. annua in turf grass consisting of high proportions of Bentgrass, or Festuca species. This is an unexpected finding for two reasons. First, P. annua is usually stated in the literature to be non- mycorrhizal or at most weakly mycorrhizal. Secondly, whilst it has now- been found that VA mycorrhizae do colonise Poa annua roots, the colonisation retards rather than encourages growth. Moreover, this effect has been found to be independent of phosphorus levels in the soil. The present invention is therefore based on the discovery of a novel technical effect of VA mycorrhizae which leads to a method of treatment 3 of turfgrass which has not been contemplated hitherto. It is to be understood that the application of VA mycorrhizae, either to grassland or soil in preparation for grass sowing, in accordance with this invention is for the specific intention of suppressing Poa annua, as distinct from any other purpose e.g. to stimulate growth or for the recovery of damaged turf. Preparations for use according to the invention do not require the presence of any other bacteria or other organisms or biologically active materials, although these are not excluded if desired as incidental to the primary purpose of the invention. It is especially important that the composition applied to turf contains very little of, and is preferably substantially free of, other biological materials which could promote growth of P. annua and therefore compete with the VA mycorrhiza and conflict with the objectives of the present invention. For example, because P. annua dominates turf in areas of high phosphate application, it is highly desirable to avoid the presence in the composition of significant amounts of bacteria which release P from phosphate. The deliberate addition of such phosphate-solubilizing bacteria to the composition is strongly contra- indicated. Preferably, therefore, apart from minimal amounts of other organisms which may be present due to the use of conditions of production which are not totally sterile, the VA mycorrhiza fungi used constitute the sole or principal organismic component of the compositions of the present invention. The present invention comprises the use of a VA mycorrhiza as a growth-retardant for Poa annua. The term 'VA mycorrhiza' is used 4 herein to cover all soil-borne fungi which form arbuscules in obligate mutualistic associations with terrestrial plants. Typical representatives of the vesicular- arbuscular fungi are found among the genera Glomus, Acaulospora, Entrophosphora, Gigaspora, Scutellospora and Sclerocytis. Exemplary strains are Glomus fasciculatum, Glomus caledonium, Glomus mosseae, Glomus versiforme, Glomus intraradices, and Glomus etunicatum. One or more strains of VA mycorrhiza may be used depending on the quality of the turfgrass to be treated. Preferred strains are those of Glomus mosseae, caledonium, fasciculatum, and versiforme. VA mycorrhizae are widely available organisms and may be obtained, for example, from the International Culture Collection of VAM fungi (INVAM), Florida, United States of America. To produce VA mycorrhizal inoculum for the purposes of the present invention all infective structures of the fungus can be used, including spores and mycelium produced inside or outside the host root. Infected roots and infected substrates can also be used. Inoculum can be used in the form of slurries, gels, pellets, infected soil, or spore formulations. Especially effective formulations are the clay carrier-based formulations containing the four preferred strains indicated above e.g. those available commercially under the Registered Trade Mark 'VAMINOC (The MicroBio Group Ltd, Whittlesford, Cambridge. UK), such as VAMINOC 8/16, VAMINOC 30/60, AND VAMINOC-T. Inoculant particle sizes can be up to 2mm or 4-8mm. Alternative carriers can be selected from, for example, silica gel, bleaching earths, pumice, bauxite, attapulgite, 5 vermiculite, calcined montmorillinite, soil, peat, sand or any other substantially chemically inert material with a suitable porous structure. The grass which may be treated according to the present invention may be a sport or amenity turf which is already established and which contains unacceptable proportions of P. annua. Turf which is mown regularly is particularly suitable for treatment. Alternatively the treatment may be applied to ground on which turf is to be laid or sown e.g. a sand/peat based green sown with bentgrass or a bentgrass/Festuca mixture. Desirably, the inoculum infectivity, as measured by the "most probable number" (MPN) technique, corresponds to MPN values of from at least 200 or 500 up to 1000 propagules or more per gram. Application of inoculum at a rate of at least 5 or 10 g/m 2 is effective but higher dosages may be used if preferred, e.g. up to about 100 to 250 g/m 2 . Even higher doses, for example up to and in excess of 1 Kg/mf, are envisaged in certain applications. The inoculum can be watered into an established green or introduced into the root zone. Application in weather which is either very warm or very cold is inadvisable. The turfgrass may be a golf green, a bowling green, a lawn, or any other kind of grassy area requiring treatment according to the invention. The invention is of particular importance in improving or maintaining the quality of turf consisting mainly of Agrostis Spp or Festuca Spp. The invention also comprises a method of controlling or reducing the amount of P. annua in turfgrass which comprises applying to the 6 turfgrass a growth-inhibiting strain of VA mycorrhiza. Preferably, a composition containing infective propagules is applied to the turfgrass, the composition containing propagules in sufficient quantities to effectively colonise plant roots. Infective propagules can be selected from, but are by no means limited to, fungal spores, mycelium, hyphae, and fragments of mycorrhizal infected roots. Experiments which have established the growth retarding activity of VA mycorrhizae are described below and illustrated in Figures 1 to 7 of the accompanying drawings. Initial Experiments In July 1997, three soil cores, each 1 .5cm diameter were taken from all 18 greens on a golf course. Each core was examined under a binocular microscope and the number of tillers of each grass species counted. Roots of the grass species were then washed free of soil and placed on microscope slides. They were then examined under an epifluorescence microscope fitted with a UV bulb, under which conditions, the arbuscules (the only definitive structure of the mycorrhiza) autofluoresce. Mycorrhizae were quantified with the cross-hair eye piece method of McGonigle et al. , 1990. Differences between greens in the abundance of AMG were tested for with Kruskall-Wallis one way Analysis of Variance and the relation between mycorrhizal colonisation and AMG abundance examined with linear regression. Laboratory Experiment 7 Eighty flower pots, each measuring 7cm x 7cm were filled with a mixture of 80% sand, 20% fen loam soil and sown with Agrostis stolonifera at the rate of 40g m '~ . Half of the pots were inoculated with 8.7g of Vaminoc-T, a four species mixture of mycorrhizal fungi (The MicroBio Group Ltd, Whittlesford, Cambridge, UK) equivalent to 25g per litre of soil. Ten mycorrhizal and ten non-mycorrhizal pots then received 0.2g of AMG (equivalent to 157 000 seeds m "~ ), ten of each treatment received 0.4g of AMG seed (314 000 seeds m ~~ ) and ten of each treatment, 0.6g of AMG seed (471 000 seeds m '" ). There were also ten pots of each mycorrhizal treatment which received no AMG seed. Therefore, in total there were eight treatments (2 levels of mycorrhiza x 4 levels of AMG), with ten replicates of each, giving 80 pots. Pots were maintained in a Constant Environment room at 20C for three months, by which time a close sward had developed. The cutting height of the grass was gradually reduced to 8mm and at each cut the clippings were removed and dried to constant weight in an oven. Fertiliser was applied to mimic that recommended for an establishing green. At the end of the experiment, the total biomass produced in each pot was calculated. The effect of adding AMG and the mycorrhiza on biomass production was analysed with a nested Analysis of Variance. Results There was an expected effect of AMG addition on total biomass, but no overall effect of mycorrhizal addition. However, there was a highly significant interaction between AMG and mycorrhiza (F 3 72 = 4.5, p < 8 0.01). This was because when no AMG was added to the pots, addition of mycorrhiza increased the biomass of the bent grass. However, in all AMG addition treatments, the mycorrhiza did not increase the total biomass, and in some cases, significantly reduced it. These data therefore imply that mycorrhiza had a positive effect on the bentgrass, but a negative effect on the AMG. This can be seen by comparing pairs of treatments in the experiment. Thus, the addition of 0.2g of AMG to non mycorrhizal pots caused an increase of 22% in total biomass, while the addition of 0.2g of AMG to mycorrhizal pots resulted in a 3% decrease in biomass. Meanwhile adding 0.6g of AMG to non- mycorrhizal pots gave a 45% increase, while adding 0.6g of AMG to mycorrhizal pots gave only a 25% increase. The results from the laboratory experiment lead to the following observations. In the 'control' situation, where no \MG was sown into the pots, the addition of mycorrhizae had a positive effect on the growth of the bentgrass. In pots where no mycorrhiza was added, then the addition of the AMG seed significantly increased biomass. as would be expected. However, if the mycorrhiza was present, addition of AMG at the level of 157 000 seeds m- 2 had no effect on the total biomass and actually resulted in a small decrease. Indeed even at the highest level of sowing (471 000 seeds m- 2 ), the mycorrhiza appeared to be able to mitigate the effects of this seed addition. The seed additions in this experiment were designed to be realistic compared with tiller densities in the field. The presence of mycorrhizal fungi may serve to maintain AMG at lower levels than would occur if the fungi were absent. Furthermore, this 9 mechanism can work at tiller densities comparable to those encountered on courses. Field Study A significant negative relation between AMG abundance and mycorrhizal colonisation was again found ( F 16 = 8.65 P < 0. 01 , r " = 0. 351). Mycorrhizal colonisation was very low, compared with natural areas, but was in the range (0 - 11 %) The mean number of AMG tillers per core was 75.9 +/_ 5.3. Agrostis stolonifera in the course studied was virtually absent. In fact, 12 of the 18 greens appeared to be entirely AMG, and the mean AMG proportion in the sward was 96.1 % . There was a significant difference between greens in the density of all grass tillers (c ~ = 29.3, P < 0.05) and also in the proportion of AMG in the sward (c " = 28.7, P < 0.05). Therefore, the greens were not providing uniform surfaces, due to the variable amounts of AMG in them. Greenhouse Trials Experiment 1. Effect of Vaminoc on growth of Poa annua 8 x 1 litre trays, each measuring 20 x 15x 5 cm were filled with 800 g of a mixture of sandy soil (kettering loam from Rothamsted Experimental Station) and sand (1 : 1 w/w). 4 trays each were inoculated with 0.7 g Vaminoc ( equivalent to 20 g / m ~~ ), a three species mixture of VA- mycorrhizal fungi (MicroBio Group Ltd. , Whittlesford, Cambridge, UK). 10 and 4 trays were left uninoculated . VAM inoculation was performed by raking Vaminoc lightly into the soil surface before seed sowing. Each tray was sown with lg of Poa annua seeds , equivalent to the recommended rate of 35 g / m "" , and maintained in a glasshouse at 25 / 20 °C day /night temperature. Grass shoots were cut 4 and 10 weeks after sowing and Shoot Fresh Weight determined. At the end of the growth period (10 weeks) the total weights were calculated in each tray and root samples were stained and visually assessed for presence of VA- mycorrhizal colonization. Results: Figure la and Figure lb (photograph) A significant reduction of growth of Poa annua in response to Vaminoc inoculation as compared to uninoculated controls was observed. The detrimental effect of VA-mycorrhizal on Poa can be seen in Figure lb, taken after 10 weeks growth, before 2nd fresh weight determination. VA-mycorrhizal colonization was present in inoculated Poa but not in uninoculated control. Experiment 2. Effect of Vaminoc on Poa annua under three P levels. It is well established that the growth response of plants to mycorrhizal infection is influenced by the amount of phosphorus (P) supplied in the soil. When P is readily available to the plant the positive growth response due to mycorrhizal infection is also reduced, and it has been reported that high soil P levels reduce percentage infection in mycorrhizal plants. 11 Since VA-mycorrhiza have a negative effect on Poa annua , an experiment to study the relation between soil P levels and VAM response in Poa was performed by testing the effect of VAM inoculation under three P levels. Materials and Methods were as indicated in Experiment 1. Three soil P levels were chosen: no additional P (0), 152 g and 308 g of TripleSuperphosphate (TSP) per tray ( equivalent to 0, 100, and 240 kg/ha respectively). Therefore, there were 6 treatments ( 2 levels of mycorrhiza x 3 TSP levels), with 4 replicates of each, giving a total of 24 trays. Shoot fresh weight was determined in each tray 4 and 9 weeks after sowing. Total weights calculated after 9 weeks were analysed using a Two-way ANOVA. At the end of the experiment root samples were stained and checked for VAM fungal colonization. The results are shown in Figure 2. As expected, the overall weights for Poa annua trays not inoculated with Vaminoc were higher than those for the inoculated trays (F L 18 = 57.37; p <0.01). There was also a difference in the overall weights for the different levels of TSP ( F 2 18 = 5.97; p= 0.01). Growth responses of Poa to the three TSP levels were different in inoculated as compared to uninoculated trays ( F ;8 = 8.87; p=0.002). In inoculated trays the growth retardant effect of Vaminoc was observed at all P levels tested. At the end of the experiment, VAM colonization in roots of all inoculated Poa treatments were observed as compared to no colonization in the uninoculated controls. Some mycorrhizal fungi are detrimentally 12 affected by high soil phosphorus levels. When soil phosphorus is abundant, some fungal species grow very poorly and plant response is also reduced. However, an important feature of the effect described above is that the antagonistic effect on Poa annua has been found at low, medium and high soil P levels. The 'high' P level in this experiment corresponded to that found in typical putting green soil, where levels are invariably high. Thus, the antagonistic effect of the fungi on P. annua occurs in realistic situations, such as those found in golf greens. EXAMPLE 1 A practice putting green was used for the experiment. This was composed of a mixture of Agrostis spp. and Poa annua, with small amounts of Festuca spp. The experiment took 6-7 months from late June onward. Twenty four plots, each 0.5m x 0.5m were laid out on the green and each plot was subjected to one of four treatments: Addition of Agrostis seed, as an overseed, @ 4g per plot Addition of Vaminoc-T, @ 5g per plot Addition of seed and inoculum Addition of neither ('control'). The plots were arranged randomly on the green, with six replicates of each treatment. Grass abundance was sampled non-destructively by the point quadrant method. Twenty 3mm diameter metal pins were placed randomly in each plot, so that the tip of each pin rested on the soil surface. The total number of touches of each grass species on each pin was counted, and summed over the 20 pins in each plot. Plots were sampled on five occasions over a six month period after the treatments 13 had been applied. The average value was then calculated across the six replicates for each treatment on each date. The averages are displayed in the accompanying figures. Results (see Figs. 3. 4. and 5) At the start of the experiment, the total amount of grass in each treatment was similar. Addition of seed had no effect on the total amount of grass, or that of the two commonest species, Agrostis and Poa. However, by the end of the study, treatments which had the Vaminoc-T mycorrhiza inoculant applied to them had significantly more total grass touches (Fig. 3). The addition of the fungi therefore had stimulatory effect on sward growth with more grass blades being touched by the 20 pins. However, the effect on the two dominant grass species was not the same. The treatments in which Vaminoc-T was applied had more touches of Agrostis at the end of the six-month period, as shown by the lines marked with triangles in Fig. 4. Therefore, the fungi were much more effective in increasing abundance of this grass than was the addition of seed. Meanwhile, at the end of the experiment, plots which received Vaminoc- T had fewer touches of Poa annua than did the plots in which no treatment was applied (Fig. 5). This was a complete reverse of the situation at the start of the experiment, when 'control' plots had the lowest Poa count. Therefore, application of Vaminoc-T to a working golf green resulted in a reduction in Poa annua abundance, an increase in Agrostis abundance and an increase in overall grass abundance. 14 EXAMPLE 2 VAM field efficacy For the purpose of this demonstration, two US PGA constructed greens were selected from a large popular golf course in Cambridgeshire - UK. The greens were seeded approximately three years previous, and managed as per standard practice. Much of the green was composed of Agrostis spp. , with a low level of Festuca spp. Poa annua was evident across much of the two selected greens. Each 500m " green was split equally into two plots. The greens were hollow-tined prior to treatment. Vaminoc-G was applied at 20g m " to one half of each green, whilst the other half remained untreated. Each green was lightly irrigated within 8 hours of Vaminoc-G application. Each green was visually assessed for P. annua incidence prior to Vaminoc-G application, and again approximately 2 months after application. Greens were managed as standard practice during the course of the trial. Results The relationship between Vaminoc-G application and incidence of Poa annua for both greens are recorded on the accompanying Figures 6 and 7. It is evident that although the incidence of P. annua generally declines in the greens over the assessment period as expected, the total percentage reduction in the Vaminoc-G treatment areas is far higher than that recorded in the non-treated areas (96% and 84% cf. 59% and 34% for green 1 and 18 respectively).

Claims: 15 CLAIMS 1. The use of V A mycorrhiza as a growth retardant for Poa annua. 2. The use according to Claim 1 , using one or more strains of VA mycorrhiza selected from genera Glomus, Acaulospora, Entrophosphora, Gigaspora, Scutellospora and Sclerocytis. 3. The use according to claim 1 , using one or more strains of the group consisting of Glomus fasciculatum, Glomus caledonium, Glomus mosseae, Glomus versiforme, Glomus intraradices and Glomus etunicatum. 4. The use according to Claim 1 , 2 or 3, applied to turfgrass. 5. The use according to Claim 4, in which the turfgrass is a golfing green. 6. The use according to claim 4, in which the turfgrass in an amenity turf. 7. Use according to Claim 4, 5, or 6, in which the turfgrass consists mainly of Agrostis Spp and/or Festuca Spp. 8. A method of controlling or reducing the growth of P. annua in turfgrass which comprises applying to the turfgrass one or more strains of VA mycorrhiza which are growth-controlling for P. annua. 9. A method according to claim 8, in which the one or more strains of VA mycorrhiza is/are selected from the group recited in claim 2 or 3. 10. A method according to claim 8 or 9, in which VA mycorrhiza is applied to the soil as a formulated product in which the VA mycorrhizae 16 constitute the principal component having biological activity on the growth of turf grass. 11. A method according to claim 10, in which the inoculum has an MPN of from about 200 to 1000 or more propagules per gram. 12. A method according to any of claims 8 to 11 , in which the mycorrhiza is formulated with a clay or other solid carrier. 13. A method according to claim 11 or 12, in which the amount of formulated product applied to the turf is from about 5 g/m " to about 1 kg/m " . 14. A method according to any of claims 8 to 13, in which the infective propagules are selected from fungal spores, mycelium, hyphae, and fragments of mycorrhiza infected roots. 15. A method for the control of the growth of P. annua in a sport or amenity turf, or other grassy area in need of such control, or an area in which high quality turf-grass is to be laid or sown, which comprises applying to the turf or other area a composition which contains one or more strains of VA mycorrhiza and is substantially free from any biological agent having direct or indirect P. annua growth-promoting activity.

Inventors: GANGE ALAN CHRISTOPHER, GANGE, ALAN, CHRISTOPHER
Applicants: GANGE ALAN CHRISTOPHER, MICROBIO GROUP LTD, GANGE, ALAN, CHRISTOPHER, THE MICROBIO GROUP LIMITED
Application year: 1999
Main classification: A01N63
IPC codes: A01G1, A01G7, A01N63, C12N1