Patent Application: US-26378002-A

Abstract:
a method of providing a refuge for caterpillar larvae , which are susceptible to plants expressing delta endotoxin of bacillus thuringiensis so as to delay and possibly prevent the susceptible species from developing resistance to the toxin . also provided is a method of cultivating paulownia trees so as to provide an effective refuge for the susceptible caterpillar larvae .

Description:
the inventor has discovered that a refuge can be provided for heliothis virescens , helicoverpa zea and other cotton pests that slows the development of resistance to bt in pests without sacrificing portions of the cotton crop or large portions of valuable land . the following described refuge option , developed by the inventor , has surprisingly managed to achieve this goal . certain basic factors must be taken into account in the development of an effective refuge . the placement and size of the structured refuge employed should be based on the existing target pest biology data ( e . g ., larval and adult movement , mating and ovipositional behavior , fecundity ). the refuge should be planted with an agronomically similar hybrid and as close as possible to , and at the same time as , the bt crop . refuge size should be adjusted or alternative control measures proposed if there is a lack of a high dose for a particular target pest . in the development of refuge options , the following considerations should be made . first , the applicability of an in - field versus external refuge for each target ( susceptible ) pest should be evaluated based on larval and adult movement , ovipositional and mating behavior . the proximity of an external refuge must be within the normal adult flight range to mating / ovipositional habitats . if there are multiple pests , the structured refuge options should be inclusive of all potential target ( susceptible ) pests . if there are regional pest considerations , the structured refuge options should reflect these considerations . therefore , structured refuge options should be designed within the feasibility and current understanding of the technology to address all of the target pest species including the pest with the greatest susceptibility to the bt toxin . in accordance with the present invention structured refuge options can be designed with a high degree of grower adoption in mind . an important consideration in designing a refuge is whether there is a single dominant pest for a particular region or locality and whether there is a high dose of toxin produced . in addition , refuge strategies designed in accordance with the present invention can take into consideration whether there are stacked genes versus single genes to control the same target pests or different target pests . resistance management strategies such as the structured refuge of the present invention can be designed with flexibility to accommodate rapidly changing technology ( e . g ., gene stacking or adding additional bt proteins ), new research data , and improved understanding of resistance management . a structured refuge designed in accordance with the present invention will provide sufficient susceptible adult insects to mate with potential bt - resistant adult insects to dilute the frequency of resistance genes . past efforts to achieve an effective refuge strategy for resistance control in pests management have led to controversy over the size , structure , and deployment of non - bt crop refuges and how they should be implemented on a regional basis . as discussed earlier , the conventionally recommended alternative resistant management options have required growers to plant 80 % of their land to bt cotton , but the remaining 20 % must be planted to non - bt cotton or , in the second option , to plant 95 % of the acreage to bt cotton , but 5 % of the acreage must be planted to non - bt cotton . both options require the grower to sacrifice a percentage of his production acreage to the effects of pest insects . the present invention provides an economical means of providing an effective refuge without the conventional large sacrifices of crop production and loss of agricultural land use . the present invention has provided a refuge which uses paulownia species of trees as a suitable host for pest insects , which attack cotton , such as heliothis virescens ( tobacco budworm ), a major caterpillar pest . plantings of the paulownia trees in a copse , which can be easily managed year to year , provides an excellent structured refuge on less acreage ( considerably less than 5 %) while increasing the number of susceptible pest insects such as lepidopteran larvae . the paulownia tree originated in china and was introduced to the u . s . as an ornamental plant . in recent years , some tree production farms have planted paulownia for commercial production but it has not found use in resistance management applications . the paulownia elongota is a rapid growing tree that will flower in the early spring . it can be readily maintained and pruned to limit it growth . according to the present invention , 95 - 99½ acres of a bt - crop are planted in a 100 acre field . the remaining ½ to 5 acres are planted with paulownia elongota trees spaced about 3 feet apart and maintained at a height of about 4 feet through routine trimming and pruning . while this is the preferred embodiment , it is within the concept of the invention to provide a paulownia refuge planted at varying distances up to 20 feet apart and having a height up to 15 feet or more . taller more widespread trees would necessarily require more acreage from the field ; and under optimum conditions be a less desirable option . however in areas of varying types of terrain where not all of the acreage is cultivatable , the spacing and height of the paulownia trees can be expected to vary from the preferred embodiment but still fall within the scope thereof . the paulownia trees can be maintained from year to year or replaced as needed . the trees can be grouped into a single area , or in any manner consistent with establishment of a structured refuge in accordance with epa guidelines 1 . fields of other sizes may be planted according to the relative percentages outlined above . a refuge prepared in accordance with the present invention can employ only 5 % of the available agricultural field although the present invention can be effective with 4 %, 3 %, 2 %, 1 %, or even as little as ½ % of the land devoted to refuge plant production . alternatively , trees can be grown up to their natural height and planted at larger spacings , but a larger refuge area will be required to support the same number of pest insects due to the lower foliage density . however , in areas with difficult terrain , it may be more advantageous to plant such a larger area as such terrain may not be suitable for bt - crop production . initial studies were conducted on the brown loam experiment station of mississippi state university in learned , miss . the treatments were paulownia elongota and nectaried cotton . each plot was planted on 38 inch row centers , 16 rows × 50 linear feet . cotton was planted at normal agronomic rates ( approximately 3 plants per row feet ). the seedlings trees were planted approximately 3 feet apart within rows . the data that was obtained from a sampling of the plot on a single day during the test period is shown in table 1 . the results for the same sampling in table 2 are in per acre terms . extensive studies were conducted , which compared untreated non - bt cotton to untreated paulownia with regards to production of lepidopteran larvae . paulownia elongata seedlings in 2 × 2 × 3 inch plastic cell trays were purchased from carolina pacific international , inc . ( lenox , ga .). on july 10 , four replicates of the two treatments ( paulownia and cotton ) were planted . prior to planting , all plots were disked equally to prepare the seedbed . the paulownia plots were 50 ft in length by 16 rows wide ( 38 inch centers ), and plants were spaced 38 inches apart within rows . paulownia seedlings were hand planted and one fertilizer tablet ( 20n - 10p - 5k ) purchased from carolina pacific international was placed approximately 2 inches to the side of the seedling . cotton plants (‘ suregrow 521r ’) were planted the same day using a vacuum planter . the cotton plants were planted at normal field spacing ( 3 to 4 plants per linear foot of row .). paulownia plots were flood irrigated for one week to prevent seedlings from dying . this is a normal practice for paulownia transplants . plants were monitored on a weekly basis for heliothine eggs and / or larvae , and other lepidopteran larvae . initial data included number of eggs and / or larvae visually observed per 25 plants . after initial observations were made , the inventor determined that the size of the larvae observed should also be considered . therefore the visual observations were modified to include the number of small ( less than ¼ inch ), medium ( ¼ - ½ inch ), and large ( greater than ½ inch ) heliothine larvae , the number of loopers ( trichoplusia ni and pseudoplusia includens ), the number of armyworms ( pseudaletia and spodoptera spp . ), and the number of other lepidopteran larvae . table 3 provides the mean ( sem ) number of heliothine eggs , small , medium , large , and total heliothine larvae , armyworm larvae , shinx eggs and larvae , and looper larvae per acre of each species of paulownia . on september 7 a measurement of height ( in inches ) was obtained from 25 plants in each plot of paulownia and cotton . the data were recorded and analyzed for the visual observations of 25 plants per plot . the data were analyzed by anova with means separated by fisher &# 39 ; s protected lsad . resulting means and standard errors were converted to a per acre basis for both the cotton and paulownia treatments . as shown in fig1 a first sampling on august 23 , showed statistically similar numbers of heliothine ( heliothis virescens and helicoverpa zea ) larvae per acre in cotton and paulownia . however , the paulownia soon began to grow rapidly . it is believed that the paulownia required an adjustment period for the transplanted root system . approximately one week later , on august 29 , the paulownia had more heliothine larvae than did the cotton ( fig1 ), a trend that remained for sample dates through september 14 . on september 20 , larval populations on a per acre basis were low and were equivalent between paulownia and cotton . cotton had more larvae per acre on september 26 although the total population density was relatively low . it is worth noting that most of the cotton planted on normal planting dates had reached maturity by september 26 , but the cotton in this study had not reached maturity due to the late planting date ( both cotton and paulowania are normally planted in early may in mississippi , kays et al . 1997 ). however , paulowania continued to attract heliothine larvae into october , which would be after defoliation of cotton in mississippi . on october 16 , no larvae were observed on cotton although larvae were still observed on paulowania . the paulownia trees continued to grow and stay green until the first frost which could mean an extra generation of larval production in the fall for the paulownia trees . during the peak larval number ( august 19 , september 7 , and september 14 ) the paulownia had 7 . 5 , 5 . 6 , and 10 . 84 times more larvae than the cotton , respectively . larvae were collected on two dates ( august 30 and september 21 ) for species identification . on august 30 , the mandibles were removed from 10 larvae and examined under a stereoscope for species separation . on september 21 , the mandibles were removed from 25 larvae and examined for species separation . data show that 40 % of the larvae on august 30 were heliothis virescens and 60 % were helicoverpa zea . data for september 21 show that 96 % of the larvae were heliothis virescens and 4 % were helicoverpa zea . the mean number of total eggs and total heliothine larvae are shown in fig2 . more eggs were recorded on cotton than on paulownia , however , more larvae were recorded on paulownia . this observation could indicate that paulowania is a better food source than cotton ( resulting in higher survival on paulownia ). paulownia did have significantly more heliothine larvae ( approximately 2 . 7 times more larvae ) when analyzed across all sample dates ( fig2 ). once the heliothine larvae data were separated into small , medium , and large heliothine larvae , the data show most heliothine larvae observed on cotton were small larvae ( fig3 ). for all sample dates , paulownia and cotton did not differ in terms of small heliothine larvae per acre , but paulownia had 6 times more medium sized heliothine larvae , and large larvae were only observed in the paulownia plots . in addition , paulownia had significantly more looper larvae and other lepitopteran larvae than the cotton ( fig3 ). there were some other lepidopteran larvae recorded on paulownia , but not on cotton . among these other lepidopteran larvae , sphinx larvae were the predominant group . although the cotton and paulownia were planted at the same time , the paulownia had outgrown the cotton by september 7 ( fig4 ). the paulownia did have an adjustment period after transplanting but soon made the adjustment and began its rapid growth . the data indicate that paulownia has a potential to produce more medium and large larvae as compared to cotton . it seems reasonable to believe this would result in higher numbers of adult moths emerging from paulownia as well . by first frost , the paulownia had exceeded 5 feet in height . preferably , the refuge can be maintained by routine pruning to maintain the height and density of the paulowania . the purpose of paulownia refuge plantings should not be to produce timber but only to serve as a refuge . alternatively management of the refuge can include routinely cutting the plants off at ground level and allowing the plants to produce regrowth . this alternative method of maintaining the refuge can require a planting that is large enough to alternate cutting half or more of the planting and allowing regrowth before cutting the remaining portion . this would prevent flowering and insure that seed are not dispersed allowing the escape of the plant species . it should be noted that paulownia has been introduced into mississippi for over 100 years and has not become a weed problem . the inventor has discovered that paulownia can be effectively used as a refuge plant for heliothine larvae as well as other lepidopteran larvae . the refuge of the present invention would exist beyond the cotton - growing season and would require a one - time planting . 1 ) epa and usda position paper on insect resistance management in bt crops , may 27 , 1999 ( minor revisions jul . 12 , 1999 ). 2 ) caprio , m . a . 1994 . bacillus thuringiensis gene deployment and resistance management in single - and multi - tactic environments . biocontrol sci . and technol ., 4 : 487 - 497 . 3 ) craig , c ., s . steward , r . luttrell , j . robbins , and g . snodgrass . 1999 . use of alternate hosts as a trap for tarnished plant bugs and a refuge for bt - susceptible tobacco budworms . pp . 1056 - 1061 . in , p . dugger and d . richter ( eds . ), proceedings , beltwide cotton conference , national cotton council , memphis , tenn . 4 ) dicke , stpehen , forestry exgtension specialist , central mississippi res . and ext . center , raymon , miss . 5 ) fischhoff , d . a . 1992 . management of lepidopteran pests with insect resistant cotton : recommended approaches , pp . 751 - 753 . in , d . j . herber ( ed . ), proceedings , 1992 beltwide cotton production and research conference , national cotton council , memphis , tenn . 6 ) gould , f . 1988 . evolutionary biology and genetically engineered crops . bio - science . 38 : 26 - 33 . 7 ) gould , f ., a . anderson , a . reynolds , l . bumgarner , and w . moar . 1995 . selection and genetic analysis of a heliothis virescens ( lepidoptera : noctuidae ) strain with high levels of resistance to bacillus thuringiensis toxins . j . econ . entomol . 88 : 1545 - 1599 . 8 ) kays , j ., d . johnson , and j . stringer . 1997 . how to produce and market paulownia . univ . of maryland , coop . ext . ser . bull . 319 . 9 ) ilsi health and environmental sciences institute ( ilsi hesi ). 1998 . an evaluation of insect resistance management in bt field corn : a science - based framework for rishk assessment and risk management . report of an expert panel . ilsi press , washington dc . 10 ) irm guide 2002 . insect resistance management : the 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brazzel . 1965 . seasonal host activity of the bollworm and tobacco budworm during 1963 in northeast mississippi . miss . state univ ., ag . exp . sta . bull . 712 16 ) snow , j . w ., j . h . hamm and j . r . brazzel . 1966 . geranium carolinianum as an early host for heliothis zea and h virescens ( lepidoptera : noctuidae ) in the southeastern united states , with notes on associated parasites . ann . entomol . soc . am . 59 : 506 - 509 . 17 ) stadelbacher , e . a ., h . m . graham , v . e . harris , j . d . lopez , j . r . phillips and s . h . roach . 1986 . heliothis populations and wild hosts plants in the southern u . s . in , theory and tactics of heliothis population management . southern coop . series bull . no . 316 , agr . exp . sta ., oklahoma state univ ., stillwater , okla .