WO2020132758A1 - Composition pesticide organique d'extraits de plantes natives chiliennes pour une utilisation en apiculture et en agronomie - Google Patents

Composition pesticide organique d'extraits de plantes natives chiliennes pour une utilisation en apiculture et en agronomie Download PDF

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Publication number
WO2020132758A1
WO2020132758A1 PCT/CL2018/050160 CL2018050160W WO2020132758A1 WO 2020132758 A1 WO2020132758 A1 WO 2020132758A1 CL 2018050160 W CL2018050160 W CL 2018050160W WO 2020132758 A1 WO2020132758 A1 WO 2020132758A1
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WO
WIPO (PCT)
Prior art keywords
organic
lavender
eucalyptus
eucayptus
globulus
Prior art date
Application number
PCT/CL2018/050160
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English (en)
Spanish (es)
Inventor
Gloria MONTENEGRO RIZZARDI
Gabriel NÚÑEZ QUIJADA
Víctor AHUMADA GUAJARDO
Original Assignee
Pontificia Universidad Catolica De Chile
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
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Application filed by Pontificia Universidad Catolica De Chile filed Critical Pontificia Universidad Catolica De Chile
Priority to PCT/CL2018/050160 priority Critical patent/WO2020132758A1/fr
Publication of WO2020132758A1 publication Critical patent/WO2020132758A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K51/00—Appliances for treating beehives or parts thereof, e.g. for cleaning or disinfecting
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/22—Lamiaceae or Labiatae [Mint family], e.g. thyme, rosemary, skullcap, selfheal, lavender, perilla, pennyroyal, peppermint or spearmint
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/28—Myrtaceae [Myrtle family], e.g. teatree or clove

Definitions

  • the invention relates to a product of natural origin with pesticidal activity, useful in agronomic applications, even to eliminate hive mites.
  • the product corresponds to a mixture of extracts and essential oils of plants.
  • the method of obtaining and its use is also protected.
  • the inventors have developed a product that combines extracts and essential oils of plants useful to fight pests, such as mites and bacteria in the agricultural field, but at the same time harmless to bees, so much so that the composition of the The invention can be used in hives, and allows controlling varroasis, a bee disease caused by varroa mites.
  • the product of the invention combines organic extracts from the aerial parts of nalca (Gunnera chilensistinctoria), cinnamon (Drymis winter ⁇ ), maqui (Ar ⁇ stotelia chilensis), tiaca (Caldcluvia paniculata) and eucalyptus (Eucayptus globulus) essential oils and of lavender (Lavandula officinalis).
  • document EP 230 1357 A2 proposes a method of control against Varroa that involves causing an olfactory confusion of the mite, so that it cannot locate the larvae of offspring in which it parasites. This is done by introducing into the hive a porous support with essential oils, which due to their strong aroma confuse the mite, and the infestation of the young is avoided.
  • essential oils that can be used are named, there are eucalyptus and lavender oils, but of different species than those used in the invention, which are also not used in the examples herein.
  • the invention refers to a composition with extracts from 6 different plants, with an acaricidal effect, that is, direct death of the Varroa mite that parasitizes bees. Accordingly, document EP 230 1357 A2 does not anticipate the invention.
  • WO20171 13032 discloses a method of obtaining Aristotelia chilensis or maqui biomass, and the use of hydroalcoholic extract of this biomass as a biocide and antioxidant, to be added to food or pharmaceutical compositions.
  • This document does not anticipate the composition of the invention, which uses phenolic extracts from Aristotelia chilensis, combined with extracts from 5 other plants, to give a product with pesticidal activity.
  • the biocidal activity of cinnamon is known, for example Zapata, N., Vargas, M StammMonsálvez, M. and Ceballos, R., (CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 71 (1): 45-51 (JANUARY- MARCH 201 1)) describe that cinnamon extracts obtained sequentially with n-hexane, acetone and methanol, have activity against the fungus Gaeumannomyces graminis var. Tritici. This document does not disclose the use of the phenolic extract, nor the combination of cinnamon extracts with other active components, as is done in the present invention.
  • the invention relates to a composition comprising a combination of plant extracts from native Chilean plants and essential oils, which has biocidal properties, and which is useful in apicultural and agronomic applications in general.
  • the composition of the invention comprises extracts from native Chilean plants, specifically from aerial parts of plants of nalca (Gunnera chilensistinctoria), of cinnamon (Drymis winter ⁇ ), of maqui (Ar ⁇ stotelia chilensis), of tiaca (Caldcluvia paniculata) and essential oils of ( Eucayptus globulus) and lavender (Lavandula officinalis).
  • composition of the invention shows excellent biocidal properties against the analyzed pathogens, bacteria and mites.
  • An advantage of the composition of the invention is that it is harmless to bees and at the same time allows to eliminate the Varroa mite from them.
  • Bee products are contributing to the consolidation of Chile as an agri-food power.
  • beekeeping not only provides products, but also bees actively participate in cross-pollination of wild and cultivated plant species. That is why it can be affirmed that if the bees disappear, there would be a serious and authentic productive and ecological disaster in our country.
  • the exposed background reveals the importance of controlling this disease, by generating innocuous products, such as organic or physical, and with high efficacy, similar or better to the currently commercialized chemicals, in order to improve beekeeping productivity, contributing thus to improve the country's economy.
  • an organic pesticidal composition comprising a combination of organic extracts from aerial parts of nalca plants (Gunnera chilensistinctoria), cinnamon (Drymis winter ⁇ ), maqui (Ar ⁇ stotelia chilensis), tiaca (Caldcluvia paniculata ) and essential oils of (Eucayptus globulus) and lavender (Lavandula officinalis), an aqueous solvent and optionally excipients or formulation aids.
  • the composition comprises between 2 to 10% of each organic extract of: nalca (Gunnera chilensistinctoria), cinnamon (Drymis winteri), maqui (Ar ⁇ stotelia chilensis) and tiaca (Caldcluvia paniculata) and between 0.01 and 0.1% of each essential oil of eucalyptus (Eucayptus globulus) and lavender (Lavandula officinalis) and between 5 and 15% of eucalyptus (Eucayptus globulus) and lavender (Lavandula officinalis) hydrolates.
  • the phenolic compounds present are: Gallic Acid, Quercitin, Caffeic Acid, Chlorogenic Acid, Luteolin, Routine, Coumaric Acid, Synapic Acid, Apigenin and Abscisic Acid.
  • the inventors determined the concentrations of these active compounds in a composition of the invention, and the values obtained are shown in the examples. For the person skilled in the art, it will be evident, since it is worked with vegetable extracts and not with commercial chemical solutions, that the concentrations of these compounds can undergo variations in their absolute values, being able to increase or decrease 50, 40, 30, 20, 1 0 or 5% with respect to the result included in the examples. All of these variations are within the scope of the present invention.
  • stage A comprises:
  • stage B comprises:
  • stage C) the extracts and essential oils and hydrolates are combined in these proportions: from 2 to 10% of each organic extract of: nalca (Gunnera chilensistinctoria), cinnamon (Drymis winteri), maqui (Ar ⁇ stotelia chilensis) and tiaca (Caldcluvia paniculata) and between 0.01 and 0.1% of each essential oil of eucalyptus (Eucayptus globulus) and of lavender (Lavandula officinalis) and between 5 and 15% of eucalyptus (Eucayptus globulus) hydrolates and lavender (Lavandula officinalis), completing the final volume in an aqueous solvent and optionally with excipients or formulation aids.
  • the composition of the invention is useful as a biocide; and it allows to control the Varroa mite in the hives being harmless to the bees. In another embodiment, it allows bacteria in the hive to be
  • composition of the invention is useful to inhibit the growth of pathogenic microorganisms Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa.
  • composition of the invention serves to prepare sanitizing formulations, or to be applied in crops, on harvested plant material, or any other agronomic application where a biocidal action harmless to bees is required.
  • Example 1 Obtaining extracts of nalca (Gunnera chilensistinctoria), cinnamon (Drymis winteri), maqui (Aristotelia chilensis), and tiaca (Caldcluvia paniculata)
  • the liquid portion obtained undergoes a process of broken evaporation and thus eliminate the alcohol present in the extract.
  • the dry material is re-macerated for a further 15 days in 70% ethanol, ratio 1: 10.
  • the extract obtained is passed through a Büchner funnel with 0.4 Whatman filter paper, using a vacuum pump.
  • the liquid portion obtained is it undergoes a process of broken evaporation.
  • the extracts obtained in both maceration processes are combined, obtaining the final extract in water.
  • Example 2 Obtaining essential oils from eucalyptus (Eucayptus globulus) and lavender (Lavandula officinalis).
  • the harvest of the plants from which the oil will be extracted is done the same day that the extraction will be carried out.
  • the extraction is carried out by steam dragging.
  • steam is passed through the plant material, where 2 kilos of plant are weighed and 2 liters of water are used as steam.
  • this steam passes through a condenser, where it is recovered in liquid form.
  • the liquid was allowed to settle, and after a period of time had passed, the water and oils separated into 2 phases. Both phases are recovered, differentiating between oils and hydrolates. This process was repeated 6 times.
  • Example 3 Obtaining the product of the invention.
  • Table 1 Composition of the invention. An analysis of the phenolic compounds present in the product of the invention was carried out by means of HPLC-MS / MS, the results are shown in Table 2.
  • the composition of the invention has a high concentration of Gallic Acid and Quercitin, in a lower proportion of Caffeic Acid, and in an even lower proportion Chlorogenic Acid, Luteolin, Routine, Coumaric Acid, Synapic Acid, Apigenin and Abscisic Acid.
  • Example 4 In vitro tests of safety on bees.
  • the extract used evaporates and moves through the plate. Distilled water was used as a negative control. For each extract tested, 5 repetitions were mounted.
  • Example 5 In vitro tests for varroa control.
  • Example 3 The treatments were carried out in the same way as in Example 3, where groups of 10 bees, this time infected by Varroa, were placed in perforated 9 cm Petri dishes, which allow the gas exchange with the medium and the renewal of air inside the plate environment, in addition to air flows for the diffusion of the extracts.
  • Each Petri dish contained a feeder in which high fructose syrup was placed and a 5x6 cm filter paper where 100 pL of the extract to be tested were placed.
  • Example 3 the plates were placed in a temperature controlled incubator stove at 35 ° C, imitating the existing temperature in a bee hive.
  • Example 6 Field test of the safety product on bees and control of Varroa in the hive.
  • a field trial was conducted to test the effectiveness of the composition of the invention in controlling varroa infestations in an infested hive.
  • the Varroasis Infestation Rate was measured in the hives, before and after applying the product.
  • the DeJong method with soapy water was used to measure the Infestation Rate. The process is to use a bottle, if possible with a wide mouth, where water and detergent (dish soap) were placed. The bottle was shaken vigorously for one minute, in this way the existing varroa detached from the bees. Then it was passed through a double sieve, where the bees are retained in the first and the mites in the second. The bees were taken and the procedure was repeated 2 more times. Then, the bees and varroas count was performed.
  • the degree of infestation was calculated using the following formula:
  • % of infestation n s of varroa collected / n s of bees in sample x100
  • the bee population was estimated, counting the frames populated by hive. This is measured to ensure that the hives were in a state of appropriate strength to run the experiment, since a hive with high vigor has a better chance of survival and is more productive. Populated frames are correlated with the vigor of the bee colony. Table 5 shows the strength of the hive measured in frames populated by the hive, the initial and final infestation rate as well as the difference between the two and the percentage of reduction in infestation by applying the composition of the invention.
  • the inventors carried out a similar study with oxalic acid, a product used for this purpose, and the control efficiency reached a total of 80%, that is, a result very similar to that obtained with the composition of the invention.
  • Example 7 Determination of biocidal activity on pathogenic bacteria.
  • Tests were carried out to determine if the composition of the invention has biocidal activity on pathogenic bacteria.
  • the effect of the composition of the invention was tested on Escherichia coli, Pseudomona aeruginosa, Staphylococcus aureus and Streptococcus pyogenes, from the Natural Products Laboratory stock.
  • the bacteria were activated one day prior to use in a soy agar culture medium. The following day, a bacterial inoculum was prepared in physiological serum at a concentration of 106 CFU, according to the McFarland scale. Different tests were carried out. 7.a. Agar Diffusion Test (Well Diffusion Agar, WDA)
  • Petri dishes were seeded as grass. Once sown, 3 pockets were made in the culture medium with a punch.
  • a drop of 10Oul of the composition of the invention was placed in each pocket and the plates were incubated in a growth chamber for a time of 48 hrs.
  • As a positive control the same procedure was performed with penicillin, using commercial sensidisks.
  • the growth inhibition halo was measured. Over time, the plates were checked and it was verified in which inhibition of the growth of the bacterium occurred, measuring the formation of a halo around the place where the drop was placed, which shows the extent of inhibition of bacterial development (growth inhibition halo). The results were compared with the antibiotic Penicillin.
  • composition of the invention inhibited the growth of the 4 bacteria analyzed, while Penicillin only inhibited two of them.
  • the culture medium in this case soy broth, was prepared according to the manufacturer's instructions. Subsequently, in a 96-well ELISA plate, 150 ml of soy broth was added to each of the wells, using a multichannel micropipette. Added Only in the first well of the plate, 150 ml of the composition of the invention obtained in Example 3.
  • ELISA plates have letters to indicate the rows (AH) and numbers to indicate the columns (1-12).
  • the tested solution was deposited in wells A1 -H1. With the help of a micropipette, serial dilutions were made from column # 1 to column # 12, removing what remains in the micropipette after the last column. Then, with the help of a repeater pipette, add 50 ml of bacterial solution to each of the wells. Finally the plates were incubated in an oven at 37 ° C for 24 hours and observe the results.
  • composition of the invention had an effect of inhibiting the growth of bacteria to well 5 (32-fold dilution) and 7 (128-fold dilution). This shows that low product concentrations can have a protective effect against bacterial growth.
  • This method is used to determine the minimum bactericidal concentration, which can be measured 24 hours after measuring the minimum inhibitory concentration, carrying out a transfer from the ELISA plate to a petri dish with fresh culture medium. If the bacteria resume their growth, it means that the tested substance only had an inhibitory effect on the growth of the bacteria, whereas, if the bacteria do not resume their growth, the tested substance had a bactericidal effect on the growth of the bacteria.
  • approximately 20 ml of Agar Müller Hinton culture medium was added to a 90 x 15 mm Petri dish. Plates were allowed to cool under hood and at room temperature for 30 minutes.
  • composition of the invention had an effect of inhibiting the growth of bacteria to well 5 (32-fold dilution) and 7 (128-fold dilution). This shows that even low concentrations of product can have a control over bacteria by eliminating them.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Pest Control & Pesticides (AREA)
  • Toxicology (AREA)
  • Botany (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne une composition pesticide organique utile dans des applications agronomiques, y compris pour éliminer des acariens de ruche, qui comprend une combinaison d'extraits organiques de nalca, cannelle, maqui et tiaca, en plus d'huiles essentielles et d'hydrolats d'eucalyptus et de lavande. L'invention concerne en outre un procédé d'obtention de ladite composition et les utilisations de celle-ci en agronomie comme composition inoffensive pour les abeilles.
PCT/CL2018/050160 2018-12-28 2018-12-28 Composition pesticide organique d'extraits de plantes natives chiliennes pour une utilisation en apiculture et en agronomie WO2020132758A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CL2018/050160 WO2020132758A1 (fr) 2018-12-28 2018-12-28 Composition pesticide organique d'extraits de plantes natives chiliennes pour une utilisation en apiculture et en agronomie

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CL2018/050160 WO2020132758A1 (fr) 2018-12-28 2018-12-28 Composition pesticide organique d'extraits de plantes natives chiliennes pour une utilisation en apiculture et en agronomie

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WO2020132758A1 true WO2020132758A1 (fr) 2020-07-02

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PCT/CL2018/050160 WO2020132758A1 (fr) 2018-12-28 2018-12-28 Composition pesticide organique d'extraits de plantes natives chiliennes pour une utilisation en apiculture et en agronomie

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007875A1 (fr) * 1989-11-27 1991-06-13 Prigli Maria Composition tuant l'acarien varroa jacobsoni parasite des abeilles
CL2016003275A1 (es) * 2016-12-20 2017-07-14 Pontificia Univ Católica De Chile Extractos de nalca (gunnera tinctoria) y tiaca (caldcluvia paniculata) con actividad antimicobacteriana y su procedimiento de obtención

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007875A1 (fr) * 1989-11-27 1991-06-13 Prigli Maria Composition tuant l'acarien varroa jacobsoni parasite des abeilles
CL2016003275A1 (es) * 2016-12-20 2017-07-14 Pontificia Univ Católica De Chile Extractos de nalca (gunnera tinctoria) y tiaca (caldcluvia paniculata) con actividad antimicobacteriana y su procedimiento de obtención

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CALDERONE N. ET AL.: "Plant Extracts for Control of the Parasitic Mite Varroa jacobsoni (Acari: Varroidae) in Colonies of the Western Honey Bee (Hymenoptera: Apidae", J. ECON. ENTOMOL., vol. 88, no. 5, 1995, pages 1211 - 1215, XP000549665 *
DAMIANI, N. ET AL.: "Acaricidal and insecticidal activity of essential oils on Varroa destructor (Acari: Varroidae) and Apis mellifera (Hymenoptera: Apidae", PARASITOL RES, vol. 106, 2009, pages 145 - 152, XP019760699, DOI: 10.1007/s00436-009-1639-y *
FIGEN KÜTÜKOĞLU , AHMET ONUR GİRİŞGİN, LEVENT AYDIN: "Varroacidal efficacies of essential oils extracted from Lavandula officinalis, Foeniculum vulgare, and Laurus nobilis in naturally infested honeybee (Apis mellifera L.) colonies", TURK. J. VET. ANIM. SCI, vol. 36, no. 5, 2012, pages 554 - 559, XP055723717 *
MUHAMMAD ABU BAKAR, MUHAMMAD ANJUM AQUEEL , ABU BAKAR MUHAMMAD RAZA , MUHAMMAD IRFAN ULLAH, MUHAMMAD ARSHAD , MUBASSHIR SOHAIL AND: "Evaluation of Few Essential Oils for the Management of Parasitic Bee Mites, Varroa destructor (Acari: Varroidae) in Apis mellifera L. Colonies", PAKISTAN J. ZOOL., vol. 49, no. 6, 2017, pages 2005 - 2010, XP055723715 *

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