Plant varieties by application of endocides

Abstract

The present invention relates generally to compositions and methods for mutating a plant and plants and plant products produced by said methods. Also, compositions and methods for controlling a plant species are disclosed herein.

Claims

1. A method of killing a plant in the order of Salviniales, the method comprising contacting the plant with a composition comprising 0.1 wt. % or more of an endocide to the plant, wherein: the endocide is 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, or a combination thereof; and contacting the plant with the endocide kills the plant.

2. The method of claim 1, wherein the endocide is 4-hydroxybenzoic acid.

3. The method of claim 1, wherein the endocide is 3,4-dihydroxybenzoic acid.

4. The method of claim 1, wherein the composition comprises both 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid.

5. The method of claim 1, wherein the composition comprises a secondary agent and/or an additional endocide to the plant other than 4 hydroxybenzoic acid or 3,4-dihydroxybenzoic acid, a derivative of the additional endocide, and/or an analogue of the additional endocide.

6. The method of claim 1, wherein the plant is Azolla caroliniana.

7. The method of claim 1, wherein the plant is Salvinia molesta.

8. The method of claim 1, wherein the composition comprises 0.125% or more by weight of the endocide.

9. The method of claim 1, wherein the composition comprises 0.25% or more by weight of the endocide.

10. The method of claim 1, wherein the composition comprises 0.5% or more by weight of the endocide.

11. The method of claim 1, wherein the composition contacts a propagule or other propagative tissue of the plant.

12. The method of claim 1, wherein the method comprises soaking a propagule or other propagative tissue of the plant in the composition for at least 7 days, 10 days, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or 12 weeks.

13. The method of claim 1, wherein the composition is applied topically to the plant, applied to a trichome of the plant, sprayed on the plant, spread around the plant, and/or dissolved in water surrounding the plant.

14. The method of claim 1, wherein contacting the plant with the composition comprises spraying the composition onto the plant, onto the ground surrounding the plant, into the ground surrounding the plant, onto water surrounding the plant, into water surrounding the plant, or a combination thereof.

15. The method of claim 1, wherein the composition further comprises glyphosate.

16. The method of claim 5, wherein the secondary agent and/or additional endocide does not naturally occur in a plant in the order of Salviniales.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

(2) FIG. 1—The diagram shows the enhanced production of endocidal camptothecins (CPTs) by “Trichome Management” techniques (e.g., decapitation pruning) or external applications of CPTs in Camptotheca trees. This abnormal morphogenesis can also be induced through extended periods of soaking the fruits in water or Camptotheca extracts. These endocide inductions or applications directly reduced apical dominance of Camptotheca, resulting in shrubbiness in Camptotheca tree.

(3) FIG. 2A-J—Lobed leaves developed in April in Liquidambar styraciflua (A. normal; B. abnormal), Moms alba (C. normal; D. abnormal), E. Quercus shumardii, F. Prunus persica, Ilex vomitoria (G. normal; H. abnormal), Elaeagnus pungens (I. normal; J. abnormal) following decapitation pruning in the previous December.

(4) FIG. 3A-D—Camptotheca lowreyana ‘Katie’ is a shrub cultivar up to 3 m in maturity (A) and has normal small leaves (C). ‘CT168’ is a dwarf cultivar developed from ‘Katie’. It can grow usually up to 1 m in maturity (B) and has fasciated stems, heterogeneous leaves, reduced internodes, and disturbed phyllotaxis (D).

(5) FIG. 4A-C—Comparison of Camptotheca lowreyana ‘Hicksii’ with its parent C. lowreyana: (A.) leaves of mature tree of C. lowreyana; (B.) leaves of mature tree of ‘Hicksii’; (C.) comparison of mature fruits of ‘Hicksii’ with three known species (upper row: left-C. acuminata and right-C. yunnanensis; bottom row: left-C. lowreyana and right-‘Hicksii’).

(6) FIGS. 5—7.32, 8.64, and 19.27% of the germinated acorns of Quercus shumardii (left), Q. texana (middle), or Q. michauxii (right) developed 2-3 stems, respectively, each after the bulk acorns soaked in relative small volume of water for about a month.

(7) FIG. 6A-D—Seedlings of Arachis hypogaea germinated from the seeds soaked in 5% EtOH extracts of A. hypogaea peanut shell for one week developed abnormal leaf morphogenesis (B-D) in comparison with normal seedlings germinated from the seeds without any treatment (A).

(8) FIG. 7—Induced pleiocotyly in Triadica sebifera by the prolonged seed soaking in water for six weeks. The photos show the normal cotyledon development (middle plant), cotyledon with two lobes (two fused cotyledons) (left), and tricotyledon (right).

(9) FIG. 8—About 18% of the germinated acorns of Quercus shumardii developed multiple stems after the acorns soaked in a treatment of 5% Q. shumardii acorn extracts.

(10) FIG. 9A-I—Abnormal leaf morphogenesis developed in the early leaves of Quercus shumardii after the acorns soaked in 5% ethanol extracts of Q. shumardii acorns (B-I) in comparison with normal leaves (A).

(11) FIG. 10A-F—Induced mutations in Brassica oleracea by soaking the seeds in a 5% solution of EtOH extracts of B. oleracea seeds for 48 h. (A) abnormal pleiocotyly (4 cotyledons). (B) leaf with two lobes. (C) leaflet on leaf. (D) normal single stem seedling without treatment. (E and F) induced shrubbiness with two more stems.

(12) FIG. 11—HPLC profiles of leaf extracts of abnormal seedling of Quercus shumardii induced by Q. shumardii acorn extracts in comparison with leaf extracts of a normal seedling and acorn extracts. The normal and bi-lobed leaves from the abnormal seedlings are similar in HPLC profiles but both had a compound (A) that was not detected in either acorns or normal seedlings of Q. shumardii.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(13) As disclosed herein, specific pruning of plants, prolonged water soaking of propagules, and extracts soaking of propagules can induce mutations. Mutations in plants may include mutations found in the entire plant or part of the plant (chimeras).

(14) Further, in newly-developed tissues in the treated plants or young seedlings developed from the treated fruits or seeds, chemical diversity of secondary metabolites are significantly enhanced. As disclosed herein, decapitation pruning of C. acuminata trees significantly induced biosynthesis of some new compounds not occurring naturally in the species. From the abnormal Q. shumardii seedlings germinated from the acorns treated by its acorn extracts (Example 5), a unique compound was found that was not detected in either acorns or normal seedlings (Example 8). This unique compound is a metabolic product of the abnormal oak seedlings.

(15) Providing some endocides at higher dosage can kill, inhibit growth, and inhibit germination or reduce germination capacity of plants; however, the endocides at lower dosage will cause mutations in plants and allow some seeds to germinate but induce mutations in germinated seedlings.

(16) 1. Mutation by Pruning

(17) As disclosed herein, specific pruning causes mutations in plants and/or new species. Such mutations and/or variations include, but are not limited to, development of coppiced plant with multiple stems (suckering) and development of leaf teeth. To date, there is no reported pruning technology suggested to develop a new plant variety, or pruning that causes mutations correlated with increased endogenous concentrations of endocides that are species specific and/or species and closely related species specific.

(18) Herein is disclosed that mutations can be induced by pruning or fragmenting.

(19) TABLE-US-00001 TABLE 1 Woody species list in the decapitation pruning or fragmenting experiments Phyllum/ Status in Kingdom Division Family Species Common Name North America Plantae dicots of Nyssaceae Camptotheca happytree cultivated Anthophyta acuminata Decaisne (angiosperms) Plantae dicots of Nyssaceae C. lowreyana Li Lowrey's cultivated Anthophyta happytree (angiosperms) Plantae dicots of Euphorbiaceae Triadica sebifera Chinese tallow invasive Anthophyta (L.) Small (angiosperms) Plantae dicots of Moraceae Morus alba L. mulberry native & Anthophyta unwanted (angiosperms) Plantae dicots of Altingiaceae Liquidambar sweetgum native & Anthophyta styraciflua L. unwanted (angiosperms) Plantae dicots of Fagaceae Q. shumardii Buckley Shumard oak native Anthophyta (angiosperms) Plantae dicots of Rosaceae Prunus persica (L.) Stoles peach tree cultivated Anthophyta (angiosperms) Plantae dicots of Aquifoliaceae Ilex vomitoria Sol. Ex yaupon native Anthophyta Alton (angiosperms) Plantae dicots of Elaeagnaceae Elaeagnus pungens thorny olive invasive Anthophyta Thunb. (angiosperms) Plantae dicots of Adoxaceae Sambucus Canadensis L. elderberry native Anthophyta (angiosperms) Plantae Pteridophyta Salviniaceae S. molesta D. S. Mitchell Giant salvinia invasive

(20) As disclosed herein, chimeras with abnormal morphogenesis were observed following decapitation pruning in C. acuminata, C. lowreyana, T. sebifera, M. alba, L. styraciflua, Q. texana, Q. shumardii, Q. michauxii, P. persica, I. vomitoria, E. pungens, O. ficus-indica, B. oleracea, A. hypogaea, S. canadensis, and/or S. molesta.

(21) It is expected that the method disclosed herein is capable of inducing mutations in a broad range of species. Further, it is expected that in some instances, an endocide induced in a plant by the methods is capable of inducing mutations in a broad range of species, in mammals, and/or in humans. It is also expected that in some instances, an endocide induced by the method is capable of inducing mutations only in the species from which the chimera was derived or also in closely related species.

(22) 2. Mutation by Soaking

(23) In current agricultural, forestry, and horticultural practices, short seed soaking in water (usually <24 h, occasionally up to several days) is recommended and prolonged soaking in water for several weeks is always avoided for optimal germination. By contrast, the inventor determined that prolonged soaking of seeds or fruits in water (several weeks) induces mutations in plants, including total mutations or partial mutations (chimeras). The mutations include, but are not limited to abnormal leaf morphogenesis in germinated seedlings similar to those induced by pruning, shrubbiness or dwarf habit, and abnormally large number of cotyledons (known as pleiocotyly or polycotyly) and/or cotyledon with two lobes (may also be interpreted as two fused cotyledons).

(24) Herein is disclosed that unconventional prolonged soaking of fruits or seeds of woody and herbaceous plants (Table 2) in water will induce mutations.

(25) TABLE-US-00002 TABLE 2 Experimental species list for unconventional prolonged soaking experiments Phyllum/ Status in Kingdom Division Family Species Common Name North America Plantae dicots of Nyssaceae Camptotheca happytree cultivated Anthophyta acuminata Decaisne (angiosperms) Plantae dicots of Nyssaceae C. lowreyana Li Lowrey's cultivated Anthophyta happytree (angiosperms) Plantae dicots of Nyssaceae C. lowreyana Li Hicks cultivated Anthophyta ‘Hicksii’ happytree (angiosperms) Plantae dicots of Fagaceae Quercus shumardii Shumard oak native Anthophyta Buckley (angiosperms) Plantae dicots of Fagaceae Q. texana Buckley Nuttall oak native Anthophyta (angiosperms) Plantae dicots of Fagaceae Q. michauxii Nuttall swamp native Anthophyta chestnut oak (angiosperms) Plantae dicots of Fabaceae Arachis hypogaea L peanut crop Anthophyta (angiosperms) Plantae dicots of Euphorbiaceae Triadica sebifera Chinese tallow invasive Anthophyta (L.) Small (angiosperms) Plantae Pteridophyta Salviniaceae S. molesta D. S. Mitchell Giant salvinia invasive

(26) Non-limiting examples disclosed herein include induction of mutation in greenhouse experiments using Camptotheca spp. Camptotheca spp. usually has no branch development in the early seedling stage. However, following the fruits soaked in water for four weeks, 15.6% C. acuminata seedlings developed 2-3 branches and 38.5% seedlings of C. lowreyana ‘Hicksii’ had 2-5 branches from the main stem (Example 3).

(27) Similar to Camptotheca, Quercus acorn soaking also induced significant abnormal morphogenesis. A prolonged soaking of bulk acorns in just enough water to cover all acorns for four weeks induced significant multiple stem development in both red oaks (Q. shumardii and Q. texana) and white oaks (e.g., Q. michauxii) (FIG. 5). The results are very similar to the short-term (48 h) soaking in acorn extracts containing endocides. Acorn endocides released from long soaking or external applied causes reduced apical dominance or shrubbiness (development of multiple stems directly from the same radicle like a suckering shrub and/or from the main stem).

(28) In another example, 10.6% of T. sebifera seedlings had pleiocotyly (3 or 4 cotyledons and cotyledons with two lobes) after the seeds were soaked in water for six weeks (FIG. 7 left and right figures compared to normal growth in middle figure).

(29) Soaking of S. molesta in water with air-dried whole plants of S. molesta is known to kill the plants (U.S. application Ser. No. 14/889,184, example 1). Herein is disclosed that prolonged soaking of dense S. molesta in limited volume of water killed some plants and induced some remaining tissues of S. molesta to develop mutations. Not to be bound by theory, it is believed that release of endocides damaged and killed some tissues and that dead and/or damaged tissues further released endocides, increasing the concentrations of endocides in the water. The concentrations of endocides killed some plants and induce some remaining tissues of S. molesta to develop mutations. Similar to Camptotheca (e.g., cultivar ‘Katie’ and ‘CT168’ with small leaves), the induced mutations in any type of S. molesta plants include multiple branches with small floating leaves. These mutations can occur after the death of all floating leaves. Similar forms of small leaves have been commonly believed to be the early growth stage of S. molesta known as the “primary stage” (vs. slightly cupped medium size floating leaves known as the “secondary stage” and larger and tightly folded floating leaves known as the “tertiary stage”). However, it is disclosed herein that the type of new growth in S. molesta primarily depends on the plant “growth stage” and the conditions under which the disturbance occurs. With no or with slight disturbance, a S. molesta plant at the “tertiary stage” develops a “tertiary” new growth, a plant at the “secondary stage” produces “secondary” new growth, and a plant at the “primary stage” has “primary” new growth only. Not to be bound by theory, after apical cuttings, it is believed that fragmenting (e.g., removing buds with or without leaves from S. molesta plants) enhance the endocide level and plants develop “secondary” or “primary” new growths. Also, if all floating leaves are killed, it is believed that endocides released by the dead tissues induces “primary” new growth from plants of any stage. Further, it is disclosed herein that some “primary” S. molesta plants remained in the “primary” stage until their death. Thus, the known “growth stages” of S. molesta are not heteroblastic development between juvenile and adulthood. Herein we name these “growth stages” as growth types: “primary stage” as type I, “secondary stage” as type II, and “tertiary stage” as type III.

(30) The type I S. molesta was induced by high level of endocides after all floating leaves were killed, the type II S. molesta was induced by moderate level of endocides after the plant was severely damaged, and type III form of new growth was produced from large plants (type III) with no or slight disturbance.

(31) In some embodiments, type II new growth was developed from fragmented buds (from type III S. molesta plants) with a few leaves (e.g. couple of pairs) or buds with a few floating leaves (e.g. couple of pairs) remained after endocide treatments (Examples 12 and 13). The experiments also show that culture of buds (physically removed from leaves) alone will lead to the development of type I growth (Example 13). Thus, not to be bound by theory, remaining living tissues may need to be poisoned by endocides to induce type I development. The endocides may be from the enhanced production in the surviving propagules following damages, dead tissues of the plants, or from external application.

(32) Some induced type I S. molesta plants might remain small in size until their death. In some embodiments, type I plants might grow into type II and even type III when the concentration of endocides decreases over time.

(33) It is expected that the method disclosed herein is capable of inducing mutations in a broad range of species. Further, it is expected that in some instances, an endocide induced in a plant by the methods is capable of inducing mutations in a broad range of species, in mammals, and/or in humans. It is also expected that in some instances, an endocide induced by the method is capable of inducing mutations only in the species from which the plant was derived or also in closely related species.

(34) 3. Mutation by Application with Endocides

(35) It is also disclosed herein that external applications of endocides to treat seeds, fruits, or other part of reproductive organs or tissues to induce mutations in plants, including total or partial mutations (chimeras). In some embodiments, the induced mutations includes mutations in the whole plant or at least one part of the plant. In some embodiments, soaking the fruits, seeds, or vegetative parts of woody and herbaceous plants (Table 3) with endocides will induce mutations.

(36) TABLE-US-00003 TABLE 3 Experimental species list for soaking experiments with endocides Phyllum/ Common Status in Kingdom Division Family Species Name North America Plantae dicots of Fabaceae Arachis hypogaea L peanut crop Anthophyta (angiosperms) Plantae dicots of Euphorbiaceae Triadica sebifera Chinese tallow invasive Anthophyta (L.) Small (angiosperms) Plantae dicots of Fagaceae Q. shumardii Buckley Shumard oak native Anthophyta (angiosperms) Plantae dicots of Brassicaceae Brassica oleracea L. broccoli crop Anthophyta (angiosperms) Plantae dicots of Cactaceae Opuntia ficus-indica nopal cactus crop Anthophyta (L.) Mill. or Indian fig (angiosperms) opuntia

(37) As non-limiting examples, a seedling A. hypogaea germinated from a seed soaked in 5% A. hypogaea nutshell extracts developed abnormal leaf morphogenesis including one or three developed leaflets (vs. normal four leaflets), lobed leaves, and flat petioles (FIG. 6B-6D compared to normal growth in FIG. 6A). For Q. shumardii, 5% ethanol extracts of acorns induced about 18% of seedlings to develop multiple stems (3-5) (FIG. 8). Further, early development leaves of almost all seedlings in the 0.5% or 5% extracts treatment group displayed abnormal morphogenesis (e.g., lobed leaves, see FIG. 9B-9I compared to normal growth in FIG. 9A).

(38) Usually, plants restored normal leaf morphogenesis in later growth. Interestingly, the abnormal morphogenesis in seedlings (both shrubbiness phenomenon and leaf shapes) caused by long soaking or external applications of acorn extracts are similar to the reduced apical dominance (coppicing and leaf shapes) following decapitation pruning of trees which also induce biosynthesis of endocides.

(39) External application of endocides also induced pleiocotyly in several species as that observed in prolonged seed soaking in water. As a non-limiting example, 13.9% of pleiocotyly was observed in T. sebifera seedlings germinated from the seeds soaked in a 5% solution of EtOH extracts from T. sebifera seeds for six weeks. Like woody species, herbaceous B. oleracea showed shrubbiness, pleiocotyly, and various abnormal leaves in seedlings germinated from seeds soaked in a 5% solution of B. oleracea seed extracts for 48 h (FIGS. 10A-10C, 10E, and 10F compared to normal growth in FIG. 10D). Of the 610 B. oleracea seedlings germinated from the 900 seeds soaked in a 5% solution of seed extracts for 48 h, approximately 1.3% developed 2-5 stems directly from the same radicle, approximately 1% had pleiocotyly, and approximately 3.5% had various abnormal leaf morphogenesis including leaves with two lobes or leaves with leaflets on surface. Following the 48 h water soaking, no seedling developed with multiple-stems or pleiocotyly and less than 0.5% of seedlings developed with an abnormal leaf. This is a lower mutation rate than the results observed in seedlings soaked in extract.

(40) External application of endocides also induced mutations in S. molesta plants. It is disclosed herein that type I and type II of S. molesta can be induced in normal (type III) S. molesta plants by endocides. The induced type of new growth in S. molesta is determined by the endocidal effects on the plant. A type II of new growth will be induced from either apical or axillary buds of type III S. molesta plants when moderate level of endocides are available, e.g., by direct application of endocides (Example 12) or enhanced production of endocides due to severe physical damages (e.g., fragmenting to remove most or all leaves from the buds) (Examples 12 and 13). However, when a higher level of endocide is available, only type I of new growth will develop from any types of S. molesta plants, e.g., enhanced production of endocides in the surviving propagules due to severe damage, endocides released after all floating leaf tissues die, external applications of high level of endocides, or both (Example 12). With no disturbance or with slight damage, type III S. molesta plants develop type III new growth only (Example 13). Some type I plants never grow into type II or type III plants, particularly when higher levels of endocides are available. 4-Hydroxybenzoic and 3,4-dihydroxybenzoic acids, two compounds isolated from S. molesta, induced such mutations in S. molesta at lower concentrations (e.g., <1%) (Example 12). However, each of these compounds alone can also eliminate some S. molesta plants, particularly at higher concentrations (>0.5%) (Example 14). At the same application dosage, the combination of these two compounds was more effective than either one alone in control of S. molesta (Examples 14 and 17). In the field trials, each of these endocidal compounds alone or in combination selectively killed large S. molesta plants (type III) in dense mats on the water surface (Example 16). In the field tests, each of these endocidal compounds alone or in combination also selectively killed S. molesta plants of type I and II on the water surface within 48 h (Example 17). It was found the endocides were more effective in killing type I or II salvinias on the water surface when combined with DAWN® dish soap (e.g., (Example 16). However, the compounds or combination with or without surfactant effectively eradicated the salvinias on the soil or the edge of water bodies (Example 17). If the level of externally applied endocides or available endocides from dead tissues is too high, all tissues will be killed and there will be no regrowth or any growth type induced. It has also been found that 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids can also effectively control Azolla but did not affect many other non-related species. However, these compounds did not induce mutation or injury in non-related species. These compounds are referred to as “salvinicides” herein.

(41) Non-limiting examples of endocides include plant matter, extracts of plant matter, and compounds such as, but not limited to:

(42) ##STR00001##
(5,6,7,8-tetramethoxycoumarin) identified from T. sebifera extract;

(43) ##STR00002##
4-hydroxybenzoic acid from S. molesta extract;

(44) ##STR00003##
3,4-dihydroxybenzoic acid from S. molesta extract;

(45) ##STR00004##
both identified from S. molesta extract;

(46) ##STR00005##
((+)-3-hydroxy-β-ionone) identified from S. molesta extract;

(47) ##STR00006##
((3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one) identified from S. molesta extract;

(48) ##STR00007##
(annuionone D) identified from S. molesta extract; and

(49) ##STR00008##
(dehydrovomifoliol) identified from S. molesta extract.

(50) As disclosed herein 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid has been shown to be an effective endocide against and can induce mutations in S. molesta and/or Azolla caroliniana. 4-Hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and some other phenolic acids were reported as the main autotoxins in tobacco (Yu, Liang et al. 2014; Yu, Shen et al. 2014) and 4-hydroxybenzoic, and other phenolic acids from root exudates of tobacco showed some inhibitory activity on growth and photosynthetic rate in tobacco (Zhang, Xu et al. 2013) (Wang, Li et al. 2014). 4-Hydroxybenzoic acid was also identified as one of the autotoxins in cowpea (Huang, Bie et al. 2010) and 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and some other phenolic acids reportedly are found in and inhibit some growth of dihuang (Li, Yang et al. 2012).

(51) However, the reported activities of these compounds are contradictory. 4-Hydroxybenzoic acid showed weaker inhibitory activity than benzoic acid on the growth of taro (Asao, Hasegawa et al. 2003) and weaker than adipic acid in growth of some leaf vegetables (Asao, Kitazawa et al. 2004). Kim et al. found that the main chemical constituents in water extracts of Phytolacca Americana leaves are phenolic compounds that include 3,4-dihydroxybenzoic acid and this extract exhibited allelopathic effects on Lactuca uindica and Sonchus oleraceus (Kim, Johnson et al. 2005). However, the extracts had little effects on seed germination of (P. americana) (Kim, Johnson et al. 2005) and extracts of two other Phytolacca species (P. esculenta and P. insularis) that contained similar level of 3,4-dihydroxybenzoic acid as P. Americana can, in contrast, slightly stimulate the germination of L. uindica and S. oleraceus at lower concentration (Kim, Johnson et al. 2005). These results indicated that 3,4-dihydroxybenzoic acid is not the active compound responsible for the phytotoxicity of P. americana extracts. Additionally, Wu et al. reported that 3,4-dihydroxybenzoic acid can increase the root mass of king protea explants at 100 mg/L but inhibit growth at 500 mg/L in MS medium culture (Wu 2006; Wu, du Toit et al. 2007). However, their data did not support the conclusion as Table 1 in both documents showed no significant difference after the 3,4-dihydroxybenzoic acid treatment at 500 mg/L in comparison with no treatment in either mean root length or fresh mass weight. Further, the king protea explants had no significant difference after treatment at 100 mg/L in comparison with no treatment in mean root length, but had significant difference in mean root fresh mass weight. Thus, the data of Wu et al. (2007) actually showed that 3,4-dihydroxybenzoic acid had no significant impacts on the root growth of king protea explants in MS medium culture.

(52) Further, reports of antimicrobial activity of phenolic acids, including 4-hydroxybenzoic acid are consistent with the growing belief that autotoxicity is primarily caused by the indirect effects of autotoxins via influencing microbes or parasitic organisms in the environment. For example, 4-hydroxybenzoic acid has been shown to decrease the Shannon-Wiener index for the rhizosphere bacterial population but increase that for the rhizosphere fungal populations (Zhou, Yu et al. 2012), stimulate the mycelial growth of Fusarium oxysporum f. sp. niveum, a fungal pathogen of watermelon (Liu, Xu et al. 2011), promote the hypha growth and spore proliferation of F. oxysporum, F. nivale, Aspergillus flavus, and A. fumigatus but also upgrade the expression of signal transduction system and nutrition metabolization related genes (Li 2012). 4-Hydroxybenzoic acid also inhibited both anthracnose pathogen and N-fixing bacteria in peanut at high concentrations (Liu, Gao et al. 2012) but stimulated the growth of Enterbacter ludwigii, a bacterial pathogen in the rhizosphere soils of Tai Zi Shen (Dai 2012).

(53) However, disclosed herein, 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid has now been shown in multiple experiments to be an effective endocide against and can induce mutations in S. molesta and/or Azolla caroliniana.

(54) The extracts described herein can be extracts made through extraction methods known in the art and combinations thereof. Non-limiting examples of extraction methods include the use of liquid-liquid extraction, solid phase extraction, aqueous extraction, ethyl acetate, alcohol, acetone, oil, supercritical carbon dioxide, heat, pressure, pressure drop extraction, ultrasonic extraction, etc. Extracts can be a liquid, solid, dried liquid, re-suspended solid, etc.

(55) It is expected that the methods disclosed herein are capable of inducing mutations in a broad range of species. Further, it is expected that in some instances, an endocide is capable of inducing mutations in a broad range of species, in mammals, and/or in humans. It is also expected that in some instances, an endocide is capable of inducing mutations only in the species from which the endocide was derived or also in closely related species.

EXAMPLES

(56) Herein is disclosed that endocides induced mutations in plants that include, but are not limited to, shrubbiness or dwarfism, pleiocotyly (multicotyledonous), abnormal leaf morphogenesis particularly leaf teeth or lobe development, and/or chemical biosynthesis and derivatization. Also disclosed herein is that 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, a derivative thereof, and/or an analogue thereof, or any combination thereof are endocides against species in the order of Salviniales, such as Salvinia molesta and Azolla caroliniana.

(57) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Mutations of Woody Plants Induced by Decapitation Pruning

(58) Mutations Induced by Decapitation

(59) Experimental Procedure: Six mature plants in the field in Nacogdoches, Tex., United States were selected from each of the nine species with various simple leaves and one species with compound leaves listed in the Table 1. The simple-leaf species include seven tree species C. acuminata, C. lowreyana, T. sebifera, M. alba, L. styraciflua, Q. shumardii, and P. persica, one shrubby species I. vomitoria, and one woody vine species E. pungens. The compound-leaf species is S. canadensis. The compound-leaf species was Sambucus Canadensis L. (Adoxaceae). Three plants from each species were served as control without any treatment. All main stems of the rest three plants from each species were removed from above ground at about 15-30 cm in December. First five newly developed leaves in each pruned plant were surveyed and photographed in March of the next year. Camptothecin (CPT) contents of the leaves in both pruned and unpruned trees were analyzed by the established method (Li et al., 2002).

(60) Results: In all treated plants of seven tree species, two shrubby species, and one woody vine species, at least one of the following mutations were observed after the pruning: serrated or lobed leaves, bifid, or trifid leaves, compound leaves (e.g., two leaflets per petiole), disturbed phyllotaxis, fasciated stems, or variegation (FIG. 2A-2J). In addition to the lobed, bifid, or trifid leaves, it was observed that S. canadensis plants produced twice pinately compound leaves with 3 leaflets (vs. normal once pinately compound leaves with 5-11 leaflets) after pruning treatment.

Example 2

Development of Lowrey's Happytree (Camptotheca lowreyana S. Y. Li) Cultivars by Unconventional Prolonged Fruit Soaking in Water

(61) General Experimental Procedure: Fruits of C. lowreyana were directly collected from a single parent tree. Randomly selected fruits were divided into two groups with 900 fruits each: The first group of fruits had no treatment and was stored at room temperature (approximately at 20° C.) for nine weeks to serve as control, and the second group of fruits was soaked in water in nine plastic containers separately (100 fruits per plastic container with 100 ml water) at room temperature for nine weeks. Both groups of fruits were then sowed in the pots with soil in greenhouse (30° C. during the day time and 20° C. at night) with daily water for germination. Weekly germination surveys were conducted and the seedlings with abnormal true leaves or stems were documented. All germinated seedlings were transported into large pots for further observation three months after germination. In the next two years, the seedlings with mutated leaves or stems were propagated from hardwood stem by cutting with rooting hormones (in a mist system in greenhouse). CPT contents of the three 3-year old plants of each developed cultivar were analyzed by the established method (Li et al., 2002).

(62) Results: In the control group, none of 422 germinated seedlings had abnormal leaves in comparison with the seedlings of C. lowreyana in field. 23 of the 69 germinated seedlings in the treatment group had mutation in at least one true leaf or stem including leaf size, lobed or bifid leaves, compound leaves (e.g., two leaflets per petiole), disturbed phyllotaxis, fasciated stems, or leaf variegation (with white and green bi-color or mosaic pattern). Two mutated seedlings were successfully propagated by cutting to produce over 150 plants each and were developed as cultivars ‘Katie’ and

(63) New Varieties Produced by Unconventional Prolonged Fruit Soaking in Water

(64) ‘Katie’: Unlike the parent C. lowreyana var. lowreyana which can grow up to 20 m in height (FIGS. 3A and 3C), ‘Katie’ is a shrub with a maximum height of 3 m (FIG. 3B). It has vigorous and dense multi-branching growth habit and smallest and lanceolate or elliptic leaves with entire margins in both juvenile and maturity stages (FIG. 3D). The cultivar has significantly higher CPTs yield with 0.4778% at average (on dry weight basis) in young leaves in comparison with its parent tree (0.3913%). It is also more hardy and drought-tolerant than natural Camptotheca taxa.

(65) ‘Hicksii’: In 1997, cultivar was developed from a shoot cutting of a C. lowreyana seedling germinated from a wild seed after prolonged soaking. tree cultivar can be distinguished by its smaller and cordate leaves with bigger teeth on margin in both juvenile and mature growth stages from its patent C. lowreyana var. lowreyana (FIGS. 4A and 4B). The average CPT concentration in young leaves of is 0.5537% (on dry weight basis) (Li, 2014). In 2014, fruits were produced from two 15-year-old trees. The morphological characteristics of the germinated seedlings are similar and consistent with the parent trees (FIG. 4C). Both vegetative and reproductive characteristics of ‘Hicksii’ can easily be distinguished from any known taxa (FIG. 4, Table 4).

(66) TABLE-US-00004 TABLE 4 Major diagnostic characters of ‘Hicksii’ from three Species of Camptotheca Major Diagnostic C. acuminata C. yunnanensis C. lowreyana C. lowreyana Characters Decaisne Dode Li ‘Hicksii’ Leaf Shape oval/ elliptic cordate/ovate cordate ovaloblong Fruit Color (dry) red brown or gray or Gray-brown or brown (RHS Color Chart) greyed-orange greyed-orange greyed-orange (200D) (167D) (164 B) (164 C) Fruit Length 22.23 ± 2.90 20.63 ± 2.03 29.73 ± 3.07 14.82 ± 4.35 (mean ± s.d., mm) Fruit Disc thick thin thin thin Thickness Fruit Surface (dry) rugose smooth & lucid smooth & lucid smooth & lucid Cotyledon Length 36.21 ± 5.81 26.92 ± 3.29 34.29 ± 4.93 22.36 ± 5.07 (mean ± s.d., mm)

Example 3

Mutations of Happytrees (Camptotheca) Induced by Unconventional Prolonged Fruit Soaking in Water

(67) General Experimental Procedure: Experimental fruits of C. acuminata and C. lowreyana ‘Hicksii’ were collected from single parent trees. 30 untreated fruits of each taxon were directly sowed in the pots in greenhouse to serve as control. 30 fruits of each taxon were soaked in a petri dish in water at room temperature (approximately at 20° C.) for 24 h and 30 fruits of each taxon were soaked under the same conditions four weeks. The soaked fruits were then sowed in the pots in greenhouse. Each of the control and soaking treatment experiments had three replications. Weekly germination surveys were conducted and the seedlings with one or more abnormal true leaves were documented. By the end of two months, the total germinated seedling number of each control or treatment and the number of seedlings with two or more stems derived directly from the fruit and branch number above the soil surface were counted.

(68) Results: For both taxa, the fruits soaked in water for 24 h had much better germination rate than either those without treatment or soaked in water for four weeks. Following the fruits soaked in water for four weeks, 15.6% C. acuminata seedlings developed 2-3 branches compared to no branch development from the fruits with 24 h of soaking or no soaking treatment and 38.5% seedlings of C. lowreyana ‘Hicksii’ had 2-5 branches from the main stem in comparison with no branch development in those germinated from the fruits with 24 h of soaking or no soaking treatment.

Example 4

Development of New Camptotheca lowreyana Variety ‘CT168’ by Pruning

(69) Experimental Procedure: During the cultivar development of ‘Katie’ (see Example 2), repeated pruning of the original mutated seedling of C. lowreyana ‘Katie’ were made to have cuttings propagated. The repeated pruning directly induced the mutation in a stem of the original seedling of ‘Katie’. The mutated stem had reduced internodes and smaller leaves. The mutated stem was propagated by cutting without hormones in the mist system in greenhouse (30° C. during the day time and 20° C. at night). In the next three years, repeated propagation by cuttings was made from the rooted plants with rooting hormones or without hormones. CPT contents of the three 3-year old plants of each developed cultivar were analyzed by the established method (Li et al., 2002).

(70) Results: Over 200 plants were propagated from the mutated stem of ‘Katie’ by cuttings. The plants propagated with hormones restored its morphological characteristics of its parent ‘Katie’. Those propagated by cuttings without any hormones had smaller heterogeneous leaves, reduced internodes, and profuse branching (FIG. 3D). This dwarf mutant, cultivar ‘CT168,’ of cultivar ‘Katie’ can grow up to 1 m only in maturity (Li, 2014) (FIG. 3B). ‘CT168’ has the highest CPT yield in young leaves among known Camptotheca taxa (0.5890%).

Example 5

Development of Shrubby Oaks (Quercus) by Unconventional Prolonged Acorn Soaking in Water

(71) General Experimental Procedure: Acorns of Q. shumardii, Q. texana, and Q. michauxii were collected from Nacogdoches, Tex., United States. Every species had 30 sound acorns in each of the following two treatments with three replications per treatment: control (no soaking treatment) and soaking in water (just adequate water to cover all acorns) for 48 h in room temperature and then continued soaking in refrigerator (4° C.) for four weeks. The acorns were sowed in the pots with Miracle Grow Potting Mix soil in greenhouse (30° C. during the day time and 20° C. at night). The seedling number with multiple stems (2-3 stems) derived directly from the same radicle in the germinated seedlings was surveyed.

(72) Results: By the end of four months, no seedlings with multiple stems were observed in the seedlings germinated from acorns without soaking treatment. For the soaked acorns, the percentage of plants developing 2-3 stems directly from the same radicle (shrubbiness) in the germinated seedlings of Q. shumardii, Q. texana, and Q. michauxii were 7.32, 8.64, and 19.27%, respectively (FIG. 5). Other mutations observed in the treated oaks were bilobed leaves, bifid leaves, and variegated leaves in a mosaic pattern.

Example 6

Mutations in Peanut (Arachis hypogaea) Induced by its Extracts and Unconventional Prolonged Soaking in Water

(73) General Experimental Procedures: The seeds of A. hypogaea were purchased from Royal Oak Peanuts/Hope & Harmony Farms, Drewryville, Va. 500 g dried pod shell and 1,500 g dried seeds (nuts) without sell were ground separately to coarse powders and extracted two times for 48 h with 95% EtOH (4.5 L and 2.5 L each, respectively) at room temperature. Extracts were evaporated under reduced pressure, and 23.4 g shell extracts and 31.2 g seed extracts were obtained. 10 g each of the EtOH extracts were dissolved and suspended in NANOPURE™ H.sub.2O and prepared separately as 200 mL experimental solution at the concentration of 5%. The seed soaking treatment experiments were conducted in NCPC Lab at room temperature. 30 A. hypogaea fruits can produce at least 0.83 g shell EtOH extracts and 12.47 g seed EtOH extracts using a ASE 2000 Accelerated Solvent Extractor (60° C., 1500 psi, 30 min static time, 100% volume flush, 120 s purge, and 2 cycles). 360 seeds in total were prepared and 30 seeds in a plastic container (14×15 cm, 0.68 L) were subjected to one of the four treatments for one week with three replications per treatment: (1) control: without any treatment and seeds were directly sowed in the pots; (2) soaked in 60 mL NANOPURE™ H.sub.2O; (3) soaked in a 60 mL 5% solution of A. hypogaea shell extracts (3 g shell extracts); and (4) soaked in a 60 mL 5% solution of A. hypogaea seed extracts (3 g seed extracts). All experimental seeds were sowed in the pots with Miracle Grow Potting Mix soil in greenhouse (30° C. during the day time and 20° C. at night). The morphological variations of each seedling were recorded weekly throughout the experimental period of three months.

(74) Results: By the end of the experiment, the seeds soaked in 5% solution of A. hypogaea seed extracts had not germinated. Four of the seven seedlings germinated from the seeds treated by 5% A. hypogaea peanut shell extracts and one of the 20 seedlings in the water soaking treatment had significant abnormal leaf development (e.g., one or three leaflets, petioleless smaller leaflets with non-entire leaf margins, or variegated leaves) and fused stems in comparison with the normal development of leaves (e.g., four leaflets, larger leaflets with entire margin) and stems among the 66 seedlings in the control (FIG. 6B-FIG. 6D compared to normal growth in FIG. 6A).

Example 7

Pleiocotyly in Chinese Tallow (Triadica sebifera) Small) Induced by its Extracts and Unconventional Prolonged Soaking in Water

(75) General Experimental Procedure: The leaves and stems of T. sebifera were collected from Nacogdoches, Tex., in October 2014 and were dried in an oven at 65° C. for 48 h. 11 kg dried leaves and stems were ground to coarse powders and each were extracted two times for 48 h with 95% EtOH (40 L and 24 L, respectively) at room temperature. Extracts were evaporated under reduced pressure. 410 g EtOH extracts were obtained and then stored in 4° C. The seeds of T. sebifera were collected from Nacogdoches, Tex., in October 2014 and were dried in an oven at 65° C. for 48 h. 110 g dried seeds were ground to coarse powders and extracted two times for 48 h with 95% EtOH (500 mL and 400 mL, respectively) at room temperature. The EtOH extracts were evaporated under reduced pressure. 6.3 g extracts were obtained and then stored in 4° C. 60 T. sebifera seeds can produce at least 2.11 g EtOH extracts using an ASE 2000 Accelerated Solvent Extractor (60° C., 1500 psi, 30 min static time, 100% volume flush, 120 s purge, and 2 cycles). Both leaf and stem extracts and seed extracts were prepared as experimental solution with NANOPURE™ H.sub.2O at 5% concentration each. A total 900 seeds were prepared for the five following treatments and each treatment included 60 seeds in petri dishes at 20° C. with three replications per treatment: (1) control: without soaking treatment, (2) water-24 h: soaked in 30 mL NANOPURE™ H.sub.2O for 24 h, (3) water-6 weeks: soaked in 30 mL NANOPURE™ H.sub.2O for six weeks, (4) 5% stem extracts-6 weeks: soaked in a 30 mL 5% solution of EtOH extracts of T. sebifera leaves and stems for six weeks, and (5) 5% seed extracts-6 weeks: soaked in a 30 mL 5% solution of EtOH extracts of T. sebifera seeds (1.5 g extracts) for six weeks. Seeds were sowed in 2-gallon pots with Miracle Grow Potting Mix soil in the greenhouse (30° C. during the day time and 20° C. at night). The number of germinated individuals and cotyledon number were recorded once every week throughout the experimental period. The germination rate and pleiocotyly rate were determined for each replicate.

(76) Results: No seedling germinated from the T. sebifera seeds treated by 5% T. sebifera leaf and stem extracts during the eight weeks of experiment. For the seeds treated in 5% T. sebifera seed extracts for six weeks, 20% were germinated and 13.9% of the seedlings were pleiocotyly (3 or 4 cotyledons and cotyledons with two lobes). For the seeds soaked in water for six weeks, the germination rate was 57.8% and 10.6% of the seedlings were pleiocotyly (3-4 cotyledons) (FIG. 7 left and right figures compared to normal growth in middle figure). The seeds soaked in water for 24 h had 28.9% germination with 1.9% pleiocotyly among the germinated seedlings. The seeds without soaking treatment had 40.6% germination and no pleiocotyly was observed in any germinated seedlings.

Example 8

Development of Mutated Shumard Oak (Quercus shumardii) Plants by Application of Shumard Oak Extracts

(77) General Experimental Procedure: Acorns of Q. shumardii were dried in an oven at 65° C. for 48 h. 1 kg dried acorns were ground to coarse powders and extracted two times for 48 h with 95% EtOH at room temperature. Extracts were evaporated under reduced pressure. 50 g EtOH extracts were obtained. The extracts were prepared as experimental solutions with NANOPURE™ H.sub.2O at 0.5 and 5% concentration, respectively. 30 Q. shumardii acorns can produce at least 18.96 g EtOH extracts using a ASE 2000 Accelerated Solvent Extractor (60° C., 1500 psi, 30 min static time, 100% volume flush, 120 s purge, and 2 cycles). A total 270 acorns were prepared for the five following treatments and each treatment included 30 acorns in a plastic container (14×15 cm, 0.68 L) at 20° C. with three replications per treatment: (1) control: no soaking treatment, (2) soaked in a 0.5% solution of Q. shumardii acorn EtOH extracts (0.75 g acorn extracts) for 48 h, and (3) soaked in a 5% solution of Q. shumardii acorn EtOH extracts (7.5 g acorn extracts) for 48 h. The acorns were then sowed in the pots with Miracle Grow Potting Mix soil in greenhouse (30° C. during the day time and 20° C. at night). Survey of seedlings was conducted three months later.

(78) Results: For Q. shumardii, 5% EtOH extracts of acorns induced about 18% of seedlings to develop multiple stems (3-5) in comparison with one stem only in the control (no soaking treatment) (see FIG. 8). Further, early leaf development leaves of almost all seedlings in the 0.5% or 5% extracts treatment group displayed abnormal morphogenesis (e.g., lobed or bifid leaves or variegated leaves (see FIG. 9B-9I)).

Example 9

Development of Mutated Broccoli (Brassica oleracea) Plants by Application of Broccoli Extracts

(79) General Experimental Procedure: The seeds of B. oleracea were dried in an oven at 65° C. for 48 h. 120 g dried seeds were ground to coarse powders and were extracted two times for 48 h each with 95% EtOH (400 mL each time) at room temperature. Extracts were evaporated under reduced pressure. 4 g EtOH extracts were obtained and then stored in 4° C. 1.5 g B. oleracea seed extracts were dissolved in NANOPURE™ H.sub.2O and prepared as 30 mL experimental solution at the concentration of 5%. 300 B. oleracea seeds can produce at least 0.1 g EtOH extracts using a ASE 2000 Accelerated Solvent Extractor (60° C., 1500 psi, 30 min static time, 100% volume flush, 120 s purge, and 2 cycles). 1,800 B. oleracea sound seeds were selected and 300 seeds in a Petri dish were subjected to one of the following soaking treatments for 48 h at room temperature with three replications per treatment: a 10 mL NANOPURE™ H.sub.2O (to serve as control) and a 10 mL 5% solution of B. oleracea seed extracts (0.5 g extracts). Seeds were sowed in germination box with Miracle Grow Potting Mix soil (50 seeds per box) in the greenhouse (30° C. during the day time and 20° C. at night). The number of germinated individuals and cotyledon number, leaf morphology, and stem number were recorded once every week throughout the 4-week experimental period.

(80) Results: Of the 610 B. oleracea seedlings germinated from the 900 seeds soaked in a 5% solution of seed extracts for 48 h, approximately 1.3% developed multiple-stems (2-5) directly from the same radicle (shrubbiness), approximately 1% had pleiocotyly (3-4 cotyledons), and approximately 3.5% had various abnormal leaf morphogenesis including leaves with two lobes or with leaflets on surfaces (FIGS. 10A-10C, 10E, and 10F compared to normal growth in FIG. 10D). In the 48 h water soaking treatment, no seedling developed with multiple-stems or pleiocotyly and less than 0.5% seedlings developed with an abnormal leaf (but significantly lower from that observed in seedlings induced by the extracts).

Example 10

Development of Muted Nopal Cactus (Opuntia ficus-indica) Plants by Application of Nopal Cactus Extracts

(81) Extraction Procedures: The fleshy oval stems (pads or paddles) of O. ficus-indica (300 g in dry weight) were ground to a coarse powder and extracted two times for 48 h each with 95% EtOH (1.2 L each time) at room temperature. The combined extracts were concentrated under reduced pressure to give 16.6 g. 5 g of extracts were dissolved in NANOPURE™ H.sub.2O and prepared as a 100 mL experimental solution at the concentration of 5% EtOH extracts of the O. ficus-indica. Six O. ficus-indica stems can produce at least 4.93 g EtOH extracts based on the above extraction experiment.

(82) Soaking Experiment: 12 pieces of O. ficus-indica stems (15-17 cm) were prepared and subjected to two treatments. Six O. ficus-indica stems were cultivated in 100 mL NANOPURE™ H.sub.2O to serve as control and six stems were cultivated in 100 mL 5% EtOH extracts of O. ficus-indica (5 g extracts) for 12 days at room temperature.

(83) Growth and Propagation Tests: Each experimental O. ficus-indica stem was placed in a one-gallon pot with Miracle Grow Potting Mix soil in the greenhouse. The living status of individuals was recorded once every week throughout the experimental period.

(84) Results: By the end of the second month, all stems of O. ficus-indica in the control group were alive and showed normal growth and development. At the same time, two of the six stems treated with O. ficus-indica extracts survived but developed larger leaves (1.5 to 2.5 cm long) and some mutated enlarged leaves were retained on new stems for several months. Usually, the leaves of O. ficus-indica are minute and are shed early in the normal development process.

Example 11

Chemical Biosynthesis and Derivatization in Mutated Leaves of Shumard Oak (Quercus shumardii) Seedlings Induced by Application of Shumard Oak Extracts

(85) General Experimental Procedures: The acorns of Q. shumardii were collected from a tree grown in Nacogdoches, Tex., United States. For acorn treatments see Example 8. One leaf was randomly collected from each of the two two-month-old normal seedlings, and one normal leaf and one bi-lobed leaf were collected from each of the two abnormal seedlings induced by 0.5% EtOH extracts of Shumard oak acorns. The samples of acorns and leaves were dried in an oven at 65° C. for 48 h. The dried samples were weighed and ground. An ASE 200 Accelerated Solvent Extractor (Dionex Corp., Sunnyvale, Calif.) was used to extract the EtOH extracts. Each of the leaf samples (0.2 g) and acorn samples (10 g) were loaded in 22 mL cells and a 33 mL cell. 95% EtOH was used as the solvent. The extraction was performed under the following parameters: 60° C., 1500 psi, 30 min static time, 100% volume flush, 120 s purge, and 1 cycle. The 95% EtOH extracts were evaporated under reduced pressure, transferred into the 10 mL volumetric flask, then diluted to volume with 95% EtOH and mixed as experimental solutions. The HPLC chromatographs of oak leaves and acorn extracts were established by Agilent 1100 HPLC system coupled to an Agilent 1100 diode array detector, and an Eclipse XDB-C18 column (4.6×150 mm, 3.5 μM) at a flow rate of 0.6 mL/min. A gradient elution was performed by using H.sub.2O (A) and CH.sub.3CN (B) as mobile phases. Elution was performed according to the following conditions: 2% B at time 0, linear increase to 98% B in 22 min, and hold 98% B for 8 min. The injection volumes were equivalent to 0.34 mg plant material for all analyses. The column temperature was maintained at 23° C. The HPLC chromatogram was standardized on retention times and peak intensities of the peaks observed at a wavelength of 254 nm.

(86) Results: The HPLC profiles of leaf samples from two normal seedlings are similar each other but significantly different from either normal or bi-lobed leaves from the abnormal seedlings induced by 0.5% EtOH extracts. The chromatographs of the extracts also showed that Q. shumardii acorns had much less chemical diversity than seedlings. Interestingly, the normal and bi-lobed leaves from the abnormal seedlings are similar in HPLC profiles but both had a compound that was not detected in either acorns or normal seedlings of Q. shumardii (FIG. 11).

Example 12

Mutations of Giant Salvinia (Salvinia molesta) Plants Induced by 4-Hydroxybenzoic and 3,4-Dihydroxybenzoic Acids Isolated from Giant Salvinia

(87) General Experimental Procedure: 4-hydroxybenzoic acid (>95%, HPLC analysis) and 3,4-dihydroxybenzoic acid (>95%) were isolated from S. molesta matter by using the method as described in Li, Wang et al. 2013. 4-Hydroxybenzoic and 3,4-dihydroxybenzoic acids were prepared as 50 mL experimental solution with NANOPURE™ water at eight concentrations, 0.015, 0.031, 0.063, 0.125, 0.25, 0.5, 1, and 2%, respectively.

(88) In each of 17 containers (14×15 cm, 0.68 L each), nine healthy and untreated living plants of S. molesta were cultured in tap water in a greenhouse (30° C. during the day time and 20° C. at night). The nine plants included three type I plants (approximately 1 g in fresh weight per plant), three type II plants (approximately 2 g per plant), and three type III plants (approximately 4 g per plant). The plants in each container were sprayed with 10 mL NANOPURE™ water or an experimental compound at various concentrations. Plant growth, morphological variation, and survival status were documented and photographed in each treatment for six weeks after the treatment. Then any new developed plants in each container were transferred into a container with new water for 10 weeks of culture observations.

(89) Results: In the control treatment with water only, the experimental S. molesta plants in both type I and II plants grew to the large sizes (type III) after six weeks of culture. The impacts of 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids on S. molesta development depend on their treatment concentrations. In the 1% 4-hydroxybenzoic acid treatment container, all six type I or II plants were dead without new growth within two days of the treatment. By the end of second week after the 1% 4-hydroxybenzoic acid treatment, three small plants (type I) emerged from the axillary buds of the type III plants after all the large floating leaves in these type III plants had died or turned brown. However, the three type I plants experienced slow growth and remained in the form of small, flat, and oval-shaped floating leaves (<10 mm in width) for the following four weeks in the original culture solution. Even after transfer of these three type I plants into another container with new water, these plants failed to turn into type II plants during the additional 10 weeks of observation. All nine S. molesta plants in the 2% 4-hydroxybenzoic acid treatment died and showed no new growth during the experiment.

(90) The application of 0.5% 4-hydroxybenzoic acid killed almost all floating leaves of the nine treated plant within a week. By the end of the second week, new floating leaves had emerged from eight plants and one type I plant had died. By the end of the six weeks of experiment, only one type III plant with partial green apical floating leaves had developed slightly cupped leaves (type II), whereas all other newly emerged plants from axillary buds remained as type I during the whole experimental period in the original culture solution. Even after transferring the eight new type I plants into new containers with new water, the growth status did not obviously improve.

(91) The 0.25% 4-hydroxybenzoic acid application killed all floating leaves of the small plants (type I and II) and partially injured floating leaves of the type III plants during the first week. Seven plants developed cupped floating leaves (type II) from apical buds of six type II or III plants and one type I plant by the end of the six weeks of experiment. Two emerged plants from the type I plants remained in the same form during the whole experimental period in the original culture solution. The number and size of floating leaves and submerged root-like leaves and internode length of all nine plants were improved after transferred into a new container.

(92) By the end of the experiment, the S. molesta plants treated by 0.125% or lower concentrations had no significant damage. Similar to those in the control group, there were only type III plants observed in these containers by the end of the six weeks of experiment and no induced type I or II plants were observed.

(93) Similar to 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid (0.25, 0.5, and 1%) also induced small leaf mutations in S. molesta. More new growth was observed following the treatments of 0.5% or 1% 3,4-dihydroxybenzoic acid than the treatments of 4-hydroxybenzoic acid, but the new plants remained as type I during the six weeks of observation. 2% 3,4-dihydroxybenzoic acid treatment killed all of the plants and no new growth was observed.

Example 13

Induction of Growth Types of Giant Salvinia (Salvinia molesta) Plants by Fragmenting

(94) General Experimental Procedure: The fragmenting experiments of S. molesta were conducted in the greenhouse (30° C. during the day time and 20° C. at night) in containers (60×43×15 cm, 27 L) with 20 L tap water. A first container was used to cultivate 30 healthy and untreated intact type III S. molesta plants, a second container was used to cultivate 30 apical buds, the fragments were cut from the healthy type III S. molesta plants by a knife blade, and a third container was used to cultivate 30 apical buds with two nodes including floating and submerged leaves, the fragments were cut from healthy type III S. molesta plants by a knife blade. Plant growth, morphological variation, and survival status were documented and photographed in each treatment three weeks after the treatment.

(95) Results: Each of the 30 type III intact S. molesta plants had type III of new growth from its terminal bud within three weeks of experiments. There were no other types of new growth or lateral stems developed in this group of plants. Seven of the 30 S. molesta buds without leaves developed type I of new growth during this period. Each of the 30 S. molesta buds with two nodes including floating and submerged leaves developed one to three axillary buds with an average of 2.97 stems (±0.62 (s.d.)) by the end of the experiment. Only type II of new growth emerged from the apical and axillary buds in this treatment.

Example 14

Elimination and Inhibition of Giant Salvinia (Salvinia molesta) and Carolina Mosquito Fern (Azolla caroliniana) Plants in Greenhouse Tests by 4-Hydroxybenzoic and 3,4-Dihydroxybenzoic Acids Isolated from Giant Salvinia

(96) General Experimental Procedure: Observations of giant salvinia growth in a greenhouse were performed under 28 different treatment conditions and control conditions with NANOPURE™ water or 0.5% DYNE-AMIC® (methyl esters of C16-C18 fatty acids, polyalkyleneoxide modified polydimethylsiloxane, and alkylphenol ethoxylate 99%, (Helena, Collierville, Tenn., United States)) (v:v).

(97) Materials—4-hydroxybenzoic acid (>95%, HPLC analysis) and 3,4-dihydroxybenzoic acid (>95%) were isolated from S. molesta matter using the method as described in (Li, Wang et al. 2013). Ethylparaben (>99%) was purchased from a commercial source. Each of 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and ethylparaben were prepared as 50 mL experimental solutions with NANOPURE™ water at the concentration of 0.5, 1.0, 1.5, and 2.0%, respectively. 0.5, 1.0, 1.5, and 2.0% 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and ethylparaben with 0.5% surfactant DYNE-AMIC® was also prepared as 50 mL experimental solutions with NANOPURE™ water. Mixtures of 4-hydroxybenzoic acid with 3,4-dihydroxybenzoic acid (2:1, w:w) were prepared as 50 mL experimental solutions with NANOPURE™ water at the total concentration of 0.5, 1.0, and 1.5%, respectively. 3,4-dihydroxybenzoic acid was mixed with ethylparaben (2:1, w:w) and prepared as 50 mL experimental solution with NANOPURE™ water at the total concentration of 0.5, 1.0, and 1.5%, respectively.

(98) Bioassay—For each treatment, a total of nine healthy and untreated living plants of S. molesta (type III, the biomass of three plants weighs approximately 10 g) were cultured in tap water in three plastic containers severing as three replications (14×15 cm, 0.68 L) with three plants each in a greenhouse (30° C. during the day time and 20° C. at night). The three plants in each of the three containers per treatment were sprayed with 10 mL NANOPURE™ water, 0.5% DYNE-AMIC®, or the experimental solution. Plant growth and survival status were documented and photographed in each treatment on day nine after the treatment. Pairwise comparisons for all treatments (including Control) in living biomass were made using Tukey test at alpha=0.05, which was done using SAS (SAS 9.4).

(99) 4-Hydroxybenzoic and 3,4-dihydroxybenzoic acids were also tested against Carolina mosquito fern (Azolla caroliniana Willd.) (family Azollaceae). Six containers (14×15 cm, 0.68 L) with full cover of A. caroliniana were included in the tests: two without any treatments as controls, two were sprayed with 10 mL 0.5% 4-hydroxybenzoic acid each, and the others were sprayed with 10 mL 0.5% 3,4-dihydroxybenzoic acid each. Plant growth and survival status were documented and photographed in each treatment on the fifth day after the treatment.

(100) Results: A summary of the results are found in Table 5. The combination of 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids showed significant inhibitive activities against S. molesta plants in comparison with the control treatment with water only. Each of 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids alone at 1% concentrations decreased the living biomass weight S. molesta by 85.5% and 76.5% nine days after the treatment, respectively, compared to the water control. The new growth from either treatment was type I only. 4-Hydroxybenzoic acid was found to be more effective than 3,4-dihydroxybenzoic acid and eliminated 100% of S. molesta plants at 2% concentrations. The mixture of 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids was more effective than either of the compounds alone in the inhibition of S. molesta. The results suggest synergistic activity when the isolated compounds are combined. The mixture of 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids at the ratio of 2:1 (w:w) killed 100% S. molesta plants at the total concentration 1% or higher. There was no new growth or induced plants observed during the experiment. Surfactant DYNE-AMIC® significantly improved the effectiveness of either isolate in the treatments. Ethylparaben had a similar role as the surfactant. 4-Hydroxybenzoic and 3,4-dihydroxybenzoic acids induced mutations of S. molesta at lower concentrations (not shown).

(101) In the Azolla experiments, the plants in the control containers grew well. Both 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids eliminated 100% of A. caroliniana at the 0.5% concentrations (not shown).

(102) TABLE-US-00005 TABLE 5 Inhibition of giant salvinia (Salvinia molesta) by 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid (means ± s.d. with the same letter do not differ significantly (p < 0.05)) Living Biomass (mean ± s.d.) (g) Total Concentrations of the Testing Compound(s) Treatments Water 0.5% 1.0% 1.5% 2.0% Water 29.8 ± 2.79 (a) DYNE-AMIC ® 20.6 ± 5.20 (ab) 4-Hydroxybenzoic 20.1 ± 4.3 ± 2.3 ± 0 acid 0.12 1.37 0.58 (f) (ab) (def) (ef) 3,4-Dihydroxybenzoic 17.1 ± 9.2 ± 14.3 ± 5.8 ± acid 1.7 7.9 12.6 10.0 (bc) (bcdef) (bcd) (cdef) Ethylparaben 18.6 ± 18.7 ± 12.9 ± 13.5 ± 2.37 1.37 4.29 0.70 (ab) (ab) (bcde) (bcde 4-Hydroxybenzoic 2.7 ± 0 0 0 acid & 2.46 (f) (f) (f) 3,4-dihydroxybenzoic (def) acid 3,4-Dihydroxybenzoic 3.1 ± 0.23 ± 0 0 acid & DYNE-AMIC ® 0.93 0.13 (f) (f) (def) (f) 4-Hydroxybenzoic 0.7 ± 0.2 ± 0.1 ± 0 acid & DYNE-AMIC ® 0.36 0.15 0.17 (f) (f) (f) (f) 3,4-Dihydroxybenzoic 12.8 ± 0.3 ± 0 0 acid & ethylparaben 8.23 0.46 (f) (f) (bcde) (f)

Example 15

Phytotoxic Analysis of 4-Hydroxybenzoic and 3,4-Dihydroxybenzoic Acids and their 14 Analogs on Giant Salvinia (Salvinia molesta)

(103) General Experimental Procedure: 4-Hydroxybenzoic and 3,4-dihydroxybenzoic acids and 14 of their analogs were purchased commercially. Each of 16 compounds were prepared as 5 mL experimental solutions with NANOPURE™ water at 1% concentration. A total of 102 healthy and untreated living type III plants of S. molesta (approximately 10 g in fresh weight each) were cultured and tested in 17 plastic containers (23×23 cm, 2.37 L) with six plants in each container in a greenhouse (30° C. during the day time and 20° C. at night). The first container served as the controls without any treatment, the plants in each of the other 16 containers were treated by one of the 16 testing compounds, respectively. The plants in each container were randomly classified into two groups evenly. For each plant in the first group, 10 μL of 1% experimental solution was applied by pipet on the upper surface of each blade of the six pairs of large floating leaves close to the terminal bud. For each plant in the second group, 10 μL of 1% experimental solution was applied by pipet on the lower surface of each blade of the six pairs of large floating leaves close to the terminal bud. The leaf surfaces were examined for damage and analyzed 72 hrs after the treatments.

(104) Results: A summary of the results is shown in Table 6. At 72 hrs after the treatments, benzoic, 2-hydroxybenzoic, 4-hydroxybenzoic, 2,3-hydroxybenzoic, 2,4-hydroxybenzoic, and 3,4-dihydroxybenzoic acids showed strong phytotoxicity (>85% for upper surface application) against S. molesta. 3,5-dihydroxybenzoic, 2,4,6-trihydroxybenzoic, 3,4,5-trihydroxybenzoic, and nicotinic acids did not show any activities on either upper or lower leaf surface applications. The remaining six compounds had moderate activities.

(105) ##STR00009##
Chemical Structure of 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid and some of their related compounds in the tests.

(106) TABLE-US-00006 TABLE 6 Inhibition of 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid and some of their related compounds against S. molesta Inhibition (%) Purity Upper leaf Lower leaf No. Compound Name (%) Application Application 1 Benzoic acid 99.5 88.89 100 2 2-Hydroxybenzoic acid 99.5 100 100 3 3-Hydroxybenzoic acid 99 36.12 66.67 4 4-Hydroxybenzoic acid 99 100 100 5 2,3-Dihydroxybenzoic 99 100 100 acid 6 2,4-Dihydroxybenzoic 99 100 72.23 acid 7 2,5-Dihydroxybenzoic 99 52.78 11.12 acid 8 2,6-Dihydroxybenzoic 98 44.44 25 acid 9 3,4-dihydroxybenzoic 97 86.12 72.23 acid 10 3,5-Dihydroxybenzoic 99 0 0 acid 11 2,3,4-Trihydroxybenzoic 98 44.45 11.12 acid 12 2,4,6-Trihydroxybenzoic 90 0 11.12 acid 13 3,4,5-Trihydroxybenzoic 99 0 0 acid 14 Phenoxyacetic acid 98 77.78 16.67 15 Isonicotinic acid 98 44.45 33.33 16 Nicotinic acid 99.5 0 0

Example 16

Inhibition of Giant Salvinia (Salvinia molesta) and Some Associated Plant Species in the Field Tests by 4-Hydroxybenzoic and 3,4-Dihydroxybenzoic Acids

(107) General Experimental Procedure: Field experiments were conducted in an isolated pond in east Texas, United States. The pond was infested with type III S. molesta plants that formed dense mats along the edge and type I and II plants floating on the water surface. 4-hydroxybenzoic and 3,4-dihydroxybenzoic acids were purchased from a commercial source (99.9%, HPLC analysis). 500 m.sup.2 of S. molesta was treated with 50 L 0.5% 4-hydroxybenzoic acid mixed with 0.25% DAWN® dish soap and 500 m.sup.2 of S. molesta was treated with 50 L 0.5% 3,4-dihydroxybenzoic acid mixed with 0.25% DAWN® dish soap by Solo 433 motorized backpack sprayer. Plant growth and survival status were documented through photographs and the living biomass was sampled 48 hrs after each treatment. The experimental species for selectivity tests were mainly common species associate with S. molesta or species growing in nearby habitats. The species included one fern species, Carolina mosquito fern (A. caroliniana), and 11 herbaceous invasive aquatic species of angiosperms (flowering plants), namely, water hyacinth (Eichharnia crassipes (Mart.) Solms) of the family Pontederiaceae, least duckweed (Lemna minuta) and Brazilian watermeal (Wolffia brasillensis Weddell) of the family Araceae, and hydrilla (Hydrilla verticillata (L.f.) Royle) of the family Hydrocharitaceae, alligator weed (Alternanthera philoxeroides Griseb.) of the family Amaranthaceae, knotweed (Polygonum sp.) and redvine (Brunnichia ovata Walter) of the family Polygonaceae, water primrose (Ludwigia sp.) of the family Onagraceae, cattail (Typha latifolia L.) of the family Typhaceae, proliferating bulrush (Isolepis prolifera (Rottb.) R. Br.) of the family Cyperaceae, and coontail (Ceratophyllum demersum L.) of the family Ceratophyllaceae and four woody plants, namely, baldcypress (Taxodium distichum (L.) Rich.) of the family Cupressaceae, loblolly pine (Pinus taeda L.) of the family Pinaceae, Chinese tallow (T. sebifera), and buttonbush (Cephalanthus occidentalis L.) of the family Rubiaceae. At least 30 plants for each of 12 herbaceous species and three seedlings of each of the four woody species were sprayed during the treatment of S. molesta.

(108) Results: >90% or >80% small S. molesta plants were found to be dead 48 h after the first treatment of either 0.5% 4-hydroxybenzoic acid or 3,4-dihydroxybenzoic acid with 0.25% soap. The new growths induced by these treatments were type I only. After the second treatments, all newly emerged type I plants were killed in either treatment. 100% of the type III S. molesta plants on the top layer of the dense mats were killed or severely injured by either compound within 48 hrs of the first treatment and some developed new growth of type II or III. These newly emerged S. molesta plants were killed or severely injured by either compound after the second treatment with some new growth of type I observed. However, none of the other species tested for selectivity except A. caroliniana were severely damaged or killed by 4-hydroxybenzoic or 3,4-dihydroxybenzoic acid after two foliar applications. These results suggest the specificity of the action of these compounds for giant salvinia.

Example 17

Inhibition of Giant Salvinia (Salvinia molesta) in the Field Tests by 4-Hydroxybenzoic and 3,4-Dihydroxybenzoic Acids

(109) General Experimental Procedure: The field experiments were conducted in isolated ponds in east Texas, United States. The ponds are small (each were 200-500 m.sup.2 in size) in the hardwood forests and each was fully covered with S. molesta plants and also included least duckweed (L. minuta) and Brazillan watermeal (W. brasillensis). The area was divided by plots, each plot had 50 m.sup.2 in area including approximately 40 m.sup.2 of type I and II S. molesta plants on the water surface and 10 m.sup.2 of type III S. molesta plants on soils. Each plot, except a control plot, was treated with 20 L one of the following experimental solutions by Solo 433 motorized backpack sprayer: 0.5% 4-hydroxybenzoic acid, 0.5% 4-hydroxybenzoic acid mixed with 0.25% DAWN® dish soap, 0.5% 4-hydroxybenzoic acid and 0.25% 3,4-dihydroxybenzoic acid, 0.5% 4-hydroxybenzoic acid and 0.25% 3,4-dihydroxybenzoic acid with 0.25% DAWN® dish soap, and 0.25% DAWN® dish soap. Each test condition had three replicates. 12 days after the treatments, plant growth status was photographed and the plant survival rate was measured by three 1×1 m random sample plots for floating plants on the water surface and two samples in 1×1 m random sample plots for plants on soils. Pairwise comparisons for all treatments (including Control) for living biomass were made using Tukey test at alpha=0.05, which was done using SAS (SAS 9.4).

(110) Results: A summary of the results are found in Table 7. 96.2% and 91.3% of S. molesta plants survived in the control plot on the water surface and the soils on the day 12 after the treatments, respectively. The type I plants continued to grow as type I form or a few became type II during the 12 day experimental period. Most of the type II plants grew but stayed in type II with some in type III. The type III plants grew only type III in the control plot. 34.7% and 14.3% of the S. molesta plants survived on the water surface and soils, respectively, following the treatment of 0.5% 4-hydroxybenzoic acid. Induced new growth in this treatment was primarily type I and II. 0.5% 4-Hydroxybenzoic with 0.25% DAWN® dish soap had similar effects on S. molesta survivals as treatment with only 0.5% 4-hydroxybenzoic acid. However, only 18.9% and 4.18% of the S. molesta plants survived on the water surface and soils, respectively, following the treatment of a combination of 0.5% 4-hydroxybenzoic acid and 0.25% 3,4-dihydroxybenzoic acid mixed with 0.25% DAWN® dish soap. The new growth following this treatment was primarily type I. In general, the two compounds or their combination killed S. molesta plants on the soils more effectively than those floating on water surface. 100% of the treated plants were dead in some spots within 48 h after treatment. 0.25% DAWN® dish soap alone had no significant impacts on S. molesta growth.

(111) TABLE-US-00007 TABLE 7 4-Hydroxybenzoic and 3,4-dihydroxybenzoic acids, two endocidal compounds effectively inhibited Salvinia molesta by the end of the 12 days after the first treatment in the field tests (means ± s.d. with the same letter do not differ significantly (p < 0.05) % of Living Biomass/Total Biomass (mean ± s.d.) Type I and II Type III Plants on Water Plants Treatments Surface on Soils Control 96.2 ± 91.3 ± 1.38 (a) 0.71 (a) 0.25% DAWN ® soap 89.2 ± 84.2 ± 0.75 (a) 5.87 (a) 0.5% 4-Hydroxybenzoic acid 34.7 ± 14.3 ± 6.81 (b) 1.06 (bc) 0.5% 4-Hydroxybenzoic acid 35.8 ± 15.5 ± with 0.25% DAWN ® soap 4.55 (b) 0.65 (b) 0.5% 4-Hydroxybenzoic acid and 18.9 ± 4.18 ± 0.25% 3,4-dihydroxybenzoic 4.91 (c) 2.52 (c) acid with 0.25% DAWN ® soap

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