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  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    Product and procedure for the control of the processionary of the pine (Thaumetopoea pityocampa) for its application for the degradation of the silk of its nests or pockets. The product consists of isolated and selected bacterial strains, with the peculiarity of degrading the silk of the nests or pockets of the processionary, accelerating its biodegradation and disorganization remarkably. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2716702A1
    申请号:ES201731407
    申请日:2017-12-14
    公开日:2019-06-14
    发明作者:Galan Alba Diez；Roman Rebeca Cobos；Perez Jose Manuel Alvarez；Garcia Sandra Gonzalez；Turienzo Jesus Esteban Medina；Garcia Mario Sanchez；Moran Miguel Angel Olego；Jimeno Jose Enrique Garzon；Sanchez Ana Maria Ibanez；Coque Juan Jose Rubio
    申请人:Rga Bio Investig S L；
    IPC主号:
    专利说明:

    [0001]
    [0002] Product and procedure for the control of the processionary of the pine (Thaumetopoea pityocampa) for its application for the degradation of the silk of its nests or pockets
    [0003]
    [0004] Product for the control of pine processionary pest and procedure for its application in forest stands. The product consists of three bacterial strains called Pseudomonas aeruginosa RGA1, Stenotrophomonas sp. RGA2 and Bacillus licheniformis RGA3.
    [0005]
    [0006] The three strains have been isolated from pine processionary silk obtained from nests collected in pine forests.
    [0007]
    [0008] These strains were selected based on their ability to biodegrade processionary silk in in vitro and in vivo trials.
    [0009]
    [0010] OBJECT OF THE INVENTION
    [0011]
    [0012] The present invention relates to the isolation of bacterial strains isolated from samples of soil, compost or pine processionary silk and their selection for their ability to biodegrade processionary silk.
    [0013]
    [0014] The object of the invention is to implement an economic technology, easy to apply and that can be applied on processionary nests colonizing forest stands of conifers in order to favor the degradation and disorganization of the processionary nests, making the plague of being more susceptible to be eliminated, either by the environmental conditions themselves, or by the application of insecticides or bioinsecticides.
    [0015]
    [0016] The method of the invention finds special application in forest stands of conifers or conifers present in recreational areas (parks and gardens) to combat the pest of processors.
    [0017]
    [0018] The isolated bacteria are capable of completely degrading processionary silk (0.25-0.5 mg / ml) in aqueous suspension. According to the method it has been found that spray application of bacterial suspensions on processionary nests accelerates from 2 to 7 times their rate of degradation, when compared with the normal rate of nest degradation under normal environmental conditions and depending on the rate of degradation of prevailing environmental conditions.
    [0019]
    [0020] BACKGROUND OF THE INVENTION
    [0021]
    [0022] The pine processionary (Thaumetopoea pityocampa) is a species of lepidopteran of the family Thaumetopoeidae . It is a very abundant species in the pine forests of Southern and Central Europe, where it is a widespread pest. In addition to the pines, it lives in other conifers such as cedars and firs. The caterpillars (larva stage) feed on needles (ho jas), causing serious damage to the tree, which may even dry up. In addition, the caterpillars are covered with stinging hairs that detach and float in the air, which can cause irritation in the ears, nose and throat in humans and other animals, as well as intense allergic reactions. The substance that confers this stinging capacity is a thermolabile toxin called Thaumatopina.
    [0023] For all these reasons, the pine processionary is considered a pest that has a great economic impact in coniferous forest exploitations, in addition to producing serious health damages in people and animals.
    [0024]
    [0025] The distribution of this species is closely related to climatic restrictions, especially with winter and summer temperatures and with the duration of daily sunlight. (Huchon & Demolin, 1970, Revue Forestiére Frangaise, (S), 220.). The range of distribution was relatively stable until the 1990s saw a drastic expansion towards the Southwest of Europe clearly associated with climate change (Battisti et al., 2005, Ecol. Appl. 15: 2084-2096), as a tendency to colonize increasing heights in mountain ranges (Battisti et al., 2006, Global Change Bio., 12: 662-671). Currently the pine transplanter is present in regions with Atlantic and Mediterranean climate in Europe (Albania, Bosnia and Herzegovina, Bulgaria, Croatia, France, Greece, Italy, Macedonia, Black Mountain, Portugal, Serbia, Slovenia, Spain and Switzerland), in part of Turkey and in North Africa (Algeria, Morocco, Tunisia and Libya) (Pimentel et al., 2010, Biological Journal of the Linnean Society, 100: 224-236)
    [0026]
    [0027] The pine processionary is present in most of Spain, only limiting its distribution in those areas where the pine area is scarce. The expansion in Spain is a real fact. Recent studies show their movements in areas where the altitude is higher. This is due to the fact that there has been an increase in the average temperature of the Iberian Peninsula between 1971 and 2000, going from 14.63 ° C to 15,09 ° C.
    [0028]
    [0029] As a consequence, foci of the pine processionary have been registered in unusual altitude conditions for these species 20 years ago (2,250 m in the Sierra Nevada, 2,200 m in the Sierra de Baza, 1,800 m in the Sierra de Gúdar and 2,000 m in the southern slopes in the Pyrenees). At present the only place where the plague has not arrived in Spain is to the Canary Islands and the last places where its presence has been detected are Ceuta and Melilla.
    [0030]
    [0031] The life cycle of the pine processionary is generally one year long, but due to the diapause that the pupae of this species can have, it can last up to four years and in this way prolong a part of the population its biological cycle.
    [0032]
    [0033] In this biological cycle we can distinguish two major stages that develop in two differentiated strategies of the forest ecosystem:
    [0034]
    [0035] • A phase that we call aerial or "epigea" on pine (phases of imago, egg and larva). It is in this phase of larva where the processionary is more harmful, since it develops a voracious appetite, consuming large amounts of plant material and when the formation of the pockets or nests of winter takes place and, therefore, the final location of the colony . In addition, after moulting the larva acquires its typical final appearance, which varies according to the climatic conditions of each zone. Also in this phase urticating darts are formed, which can cause serious allergies in humans and hives.
    [0036]
    [0037] • Another underground phase or "hypogea" (chrysalis or pupa stage) (Demolin, 1987, Etudes vauclausienne 3: 157-173). The passage from one stage to another is characterized by a pri maveral and collective migration of the caterpillars that, already mature, leave the nest to go to be buried a few centimeters underground, and later in summer by the emergence of the imagos that ensure the return on the plant host.
    [0038]
    [0039] At present there are different treatments for the control of the processionary, although they are inefficient and in some cases difficult to apply or expensive. Among these means we can mention: 1. - Chemical control. The chemical fight consists of fumigation with authorized insecticides (pyrethroids and inhibitors of chitin, avermectin, etc.). They should be applied on the larval stages in the first stages of development, before they develop the third stage stinging hairs. They can be applied using terrestrial means. Until September 15, 2012 the aerial means were used, but these have been banned in the EU, and in Spain since that date, after the publication of Royal Decree 1311/2012, of September 14, by which it is established the framework of action to achieve a sustainable use of phytosanitary products. This supposes a practical impossibility to fight effectively the processionary control in forest masses using chemical methods.
    [0040] 2. - Control with manual means. The physical means go through the elimination of the pockets, when the caterpillars are inside. These are cut, stacked and incinerated by destroying the larval population. In the past, shotguns were fired from bottom to top placing the shotgun in a direction parallel to the trunk so as not to damage the guide using very small loss cartridges. At the moment ecotramps exist that are placed around the trunk of the tree and that capture a part of the caterpillars that descend from the glasses of the trees. This method has the disadvantage that it is expensive especially for the labor cost involved, so in practice its use in forest stands is unfeasible.
    [0041]
    [0042] 3. - Biological control. It is based on the use of natural predators, mainly bats and passerines, such as the common saithe or the common cuckoo. However, it is a control method that is barely used because of the difficulty in settling the predators introduced in the forests. Another type of biological control is based on the use of Bacillus thuringiensis (Battisti et al., 1998, Journal Pest Science 71: 72-76).
    [0043]
    [0044] Therefore and by way of summary we can say that the plague of the processionary is in full expansion due to the absence of effective control methods or the prohibition of methods of application, such as the use of aerial spraying.
    [0045] Processionary silk is a highly unknown material in terms of its properties. It is a p-protein from the group of fibroins that typically has a great resistance to biodegradation (Shaw and Smith, 1961, Biochimica et Biophysica Acta 46: 302-310), when compared with the silk of other insects. , especially that produced industrially by the Bombyx mori silkworm. The nests made with this silk are highly resistant (in fact, no reference has been found in the bibliography regarding the degradation of processionary silk) and they offer shelter to the larvae, protecting them from environmental conditions (mainly low temperatures) and assuming a physical impediment to the entry of insecticides. That is why any strategy of disorganization of nests is a possible mechanism for controlling this pest.
    [0046]
    [0047] DESCRIPTION OF THE INVENTION.
    [0048]
    [0049] The technology that is recommended is designed to help solve a specific point of the problem described above, specifically to favor the degradation and disorganization of the processionary nests by applying selected bacteria with the ability to degrade processionary silk.
    [0050] A) .- Strains Pseudomonas aeruginosa RGA1, Stenotrophomonas sp. RGA2: description and Baci llus licheniformis RGA3.
    [0051]
    [0052] In one aspect the invention relates to three bacterial strains belonging to the species Pseudomonas aeruginosa, Stenotrophomonas sp. and Bacillus licheniformis selected from silk samples of processionary. These strains show a high degradation capacity of processionary silk.
    [0053] The respective cultures of the strains Pseudomonas aeruginosa RGA1, Stenotrophomonas. sp. RGA2 and Bacillus licheniformis RGA3 are conserved and permanently deposited in the Spanish Tiupo Culture Collection (CECT), located at the University of Valencia; Research Building; Burjassot Campus; 46100-Burjassot (Valencia), with the respective access numbers CeCT 9473, CECT 9474 and CECT 9493.
    [0054]
    [0055] Strains P. aeruginosa RGA1, Stenotrophomonas sp. RGA2 and Bacillus licheniformis RGA3 present, among others, the following characteristics that are indicated below:
    [0056]
    [0057] I) .- RGA1 strain. Strain RGA1 is an aerobic and Gram-negative, mobile bacillus. It shows a good growth in the range 30-37 ° C in several culture media including YPD, TSA or nutritive agar. In nutritive agar grows forming rough colonies with high and sunken center and is triad at the edges (in the form of fried egg), pigmented blue-greenish and turn the color of the medium from a yellowish color to a light brown tone. The colonies have a size of approximately 2 mm in diameter after 24 hours of incubation at 37 ° C. The partial sequencing of its 16S ribosomal DNA showed 100% homology with several strains of the species Pseudomonas aeruginosa. The sequencing of its genome showed that the most similar genomes correspond to several strains of the same species. For this reason the strain RGA1 was identified as belonging to the species Psedomonas aeruginosa.
    [0058]
    [0059] II) .- Strain RGA2. Strain RGA2 is an aerobic and Gram-negative, mobile bacillus. It presents a good growth in the range 30-37 ° C in several culture media including YPD, TSA or nutritive agar. In nutritious agar medium grows forming small circular colonies of yellow color filled with the high center. The appearance of the colonies is gelatinous and its approximate size is 1 mm after 48 of incubation at 37 ° C. The partial sequencing of its 16S ribosomal DNA showed a 100% homology with several strains of the species Stenotrophomonas sp. The sequencing of its genome showed that the most similar genomes correspond to several cepas of the same species isolated from environmental samples. For this reason, the RGA2 strain was identified as belonging to the genus Stenotrophomonas sp., Without being able to be assigned to a specific species, given the limited knowledge of this genus.
    [0060]
    [0061] NI) .- RGA3 strain. The RGA3 strain is an aerobic, sporulated and Gram-positive, mobile bacillus. It showed good growth in the range 30-37 ° C in several culture media including YPD, TSA or nutritive agar. In nutritive agar medium grows forming flat colonies of 2-4 mm in diameter after 48 hours of incubation at 37 ° C. The colonies have irregular contours, ivory or whitish tones and a rough and rough appearance in the most central and humid part and mucous at the edges. The partial sequencing of its 16S ribosomal DNA showed a homo- logy of 99% with several species of the genus Bacillus, while the sequencing of its genome showed its enormous similarity with the species Bacillus licheniformis, so this strain was identified as Bacillus licheniformis RGA3 .
    [0062]
    [0063] B) .- Isolation of the strains Pseudomonas aeruginosa RGA1, Stenotrophomonas sp. RGA2 and Bacillus licheniformis RGA3
    [0064]
    [0065] The mentioned strains have been isolated and selected from among the natural population of colonizing bacteria of processionary silk nests. These strains were isolated from silk samples obtained from old processionary nests (more than 3 months after their appearance in the tree). The silk was isolated from the nests in aseptic conditions by manual elimination of the plant material and residues of caterpillars (mainly fecal material) through its carding with a metallic comb with fine tines. A silk suspension was then prepared in sterile containers containing silk (4 mg / ml), yeast extract 0.2% and trace element mixture (ZnCL: 0.08 mg / l, FeCl3.6H2O: 0.4 mg / l, CuCl2. 2H2O: 0.02 mg / l; MnCl2.4H2O: 0.02 mg / l; Na2B4O7.10H2O: 0.02 mg / l and (NH4) aMo7O24.4H2O: 0.02 mg / l). The preparations were incubated at 25 ° C, 30 ° C and 37 ° C. Once a clear silk degradation began to be observed visually, and turbidity due to the growth of microorganisms in some tubes, serial dilutions of the cultures were performed in sterile saline solution (NaCl 0.9%). Dilutions were plated on nutrient agar plates which were incubated at 25 ° C, 30 ° C and 37 ° C for the isolation of all bacteria present. The different isolated bacteria were selected based on readily observable morphological data of the colonies (size, color, shape, type of edge, etc), as well as on the basis of their microscopic observation (cell shape and apparent mobility). All the macro and microscopically different bacteria were isolated in pure culture that were conserved in plates of nutritious agar to 4 ° C for their later analysis.
    [0066]
    [0067] C) .- Selection of strains Pseudomonas aeruginosa RGA1, Stenotrophomonas sp. RGA2 and Bacillus licheniformis RGA3.
    [0068]
    [0069] The mentioned strains were isolated and selected from a total of 22 strains isolated from silk cultures, being checked by carrying out pure cultures that had the ability to degrade silk.
    [0070] The strains RGA1, RGA2 and RGA3 can be used, individually or jointly, in different combinations, for application to processionary nests in forest stands to favor their degradation. This would contribute to the control of the processionary plague in forest maas.
    [0071]
    [0072] In addition, they can be used for in vitro degradation , in aqueous solutions, of processionary silk in order to obtain amino acids or peptides derived from their degradation.
    [0073]
    [0074] DESCRIPTION OF THE DRAWINGS
    [0075]
    [0076] To complement the description that will be made below and in order to help a better understanding of the characteristics of the invention, according to a preferred example of practical realization thereof, an integral set of said description is accompanied drawing where illustrative and non-limiting, the following has been represented:
    [0077]
    [0078] Figure 1 - Shows a graph corresponding to the evolution of the processionary silk degradation level over time carried out by the RGA1, RGA2 and RGA3 bacteria, compared to the absence of degradation observed in two negative controls (Negative control 1 -CN1- or silk incubated in the absence of microorganisms and negative Control 2 -CN2- or silk incubated in the presence of a non-degrading silk microorganism). The level of silk degradation was estimated based on the amount of amino acid leucine released. The cultures were incubated at 37 ° C.
    [0079]
    [0080] Figure 2 .- Shows the evolution of silk processionary degradation by the bacterial action verified by observation with scanning electron microscope. The figure shows the silk appearance before the application of the treatment with the RGA2 bacteria (Day 0) and after 5 days and 7 days after the application of the bacteria. We can see how the untreated silk has a homogeneous appearance, which deteriorates after 5 days of bacterial application (the filaments appear deteriorated and with obvious symptoms of degradation: irregular contours, hollows and areas of degradation). At 7 days after the application of the bacteria, the appearance of the silk fibers is totally amorphous, presenting a totally irregular and unstructured appearance. The application of RGA1 and RGA3 bacteria has a similar effect on silk, although the kinetics of degradation is different for each strain, as it can be easily deduced from the observation in Figure 1.
    [0081]
    [0082] EXAMPLES OF PREFERRED EMBODIMENT 5 OF THE INVENTION
    [0083] The following examples illustrate the invention, although they should not be considered as limiting the scope thereof.
    [0084] * Example 1. Obtaining and characterization of strains RGA1, RGA2 and RGA3.
    [0085] A total of 22 bacterial strains were isolated from silk cultures and differentiated according to macro morphological features (size, color, shape, appearance and type of border of the colony) and microscopic (cell shape and apparent mobility). easily appreciable, as well as based on other characteristics such as the production of diffusible pigments. All these strains were tested for their ability to degrade processionary silk. For this, cultures in liquid medium that incorporated a known quantity of silk were used. All the strains were incubated under these conditions at 25, 30 and 37 ° C without agitation for a period of 45 days, visually determining their capacity for silk degradation based on the disappearance and / or solubilization of silk in the culture. . Of the total number of bacteria tested, 3 were selected for their ability to make the crop silk disappear (degrade). Initially the strains, called r G a 1, RGA 2 and r G a 3 were identified by sequencing their 16S ribosomal RNA and subsequent sequencing of their genome.
    [0086] * Example 2. Biomass production of strains RGA1, RGA2 and RGA3.
    [0087] Two different methodologies were used in order to test the versatility of each strain when producing biomass.
    [0088] 2.1) .- Biomass production by growth in flasks in aerobic conditions.
    [0089] To this end, 1L flasks containing 0.2 L of medium nutritive broth were used. Each sample was inoculated with 5 ml of a culture developed in the same OD 1 medium estimated spectrophotometrically at 600 nm. The flasks were incubated at 30 ° C with constant agitation of 220 rpm in an orbital incubator. The growth was determined every 24 hours. During the process, the homogeneity of the crop was checked by direct observation under an optical microscope. At the end of the incubation, cell viability was checked by counting colony forming units per ml (CFU / ml). Both strains grew very rapidly under these conditions obtaining a large amount of biomass and a viability equal to or greater than that of the species Escherichia coli used as a control.
    [0090] 2.2) .- Production of biomass in a fermentor.
    [0091] The study was carried out in a 2.5 L Biostat-A laboratory fermenter, controlling the different parameters that affect the strains such as dissolved O2, pH, foam, acidity and temperature. For the development of the cultures, 1.5 L of nutritious medium broth was used, which was inoculated with a preculture of each bacterium until obtaining a cellular suspension of OD = 0.1 (determined at 600 nm). The pH of the culture was kept constant at a value of 7.0 and the incubation temperature was 30 ° C. Finally, the concentration of dissolved oxygen remained constant at 35%. Growth and cell viability were monitored as indicated in the previous section. Under these conditions all the strains showed a fast and homogeneous biomass production with a high cellular viability, of the same order of magnitude as the Escherichia coli strain used as a control.
    [0092] * Example 3.- In vitro tests Processionary silk degradation in cultures in medium liquid
    [0093]
    [0094] The silk degradation in cultures in liquid medium was carried out in tubes containing one of their silk beds (2.5 mg / ml) in a solution containing K2HPO4, 40mM M; KH2PO4, 22 mM; MgSO 4, 0.8 mM; sodium citrate, 1.6 mM; KNO3, 7.9 mM; sodium succinate, 19mM and trace element mixture (ZnCl2: 0.08 mg / l; FeCh.6H2O: 0.4 mg / l; CuCL.2H2O: 0.02 mg / l; MnCl2.4H2O: 0.02 mg / l; Na2B4O7.10H2O: 0.02 mg / l; ly (NH4) 6Mo7O24.4H2O: 0.02 mg / l). The cultures were incubated at 30 ° and 37 ° C and the silk degradation was monitored visually and by measuring the release of amino acids to the culture medium in reaction with ninhydrin. The results, obtained at the temperature of 37 ° C, can be seen in Figure 1. We can observe how there is an evident degradation of silk, deduced from the release of amino acids to the culture medium, as well as from visual observation, the kinetics of degradation being different for each of the isolated bacteria.
    [0095]
    [0096] * Example 4.- In vivo tests : degradation of processionary nests.
    [0097]
    [0098] Processionary nests were collected and sprayed with a suspension of the RGA1, RGA2 and RGA3 bacteria containing 107 cells per ml in nutrient broth. The nests were placed inside a plastic tray and kept in an open terrace, subject to variations in ambient temperatures. The treatment was repeated 48 hours later. The degradation of the nests was followed visually, being able to check the apparent degradation of the silk visually in all cases. The first symptoms were observed around day 6 with the RGA3 bacteria, while evident symptoms of degradation began to be observed from day 9 in the case of the RGA2 bacteria and 11 in the case of the RGA1 bacteria. The existence of an evident degradation of the silk was confirmed by electronic microscopy as can be seen in Figure 2. However, the observed kinetics of silk degradation is variable depending on environmental conditions, such as day and night temperatures and humidity. In general, mild temperatures and high environmental humidity accelerate the process.
    权利要求:
    Claims (6)
    [1]
    1. Product for the control of the processionary of the pine (Thaumetopoea pityocampa) for its application for the degradation of the silk of its nests or pockets, characterized because they are isolated and selected bacteria with capacity to degrade the silk of the processionary.
    [2]
    2. Product for the control of the processionary of the pine (Thaumetopoea pityocampa) for its application for the degradation of the silk of its nests or pockets, characterized according to the previous claim, because it is materialized in the Pseudomonas aeruginosa RGA1 strain and deposited in the CECT with the access number CECT 9473.
    [3]
    3. Product for the control of the processionary of the pine (Thaumetopoea pityocampa) for its application for the degradation of the silk of its nests or pockets, characterized according to the first claim, because it materializes in the strain Stenotrophomonas sp. RGA2 and deposited in the CECT with the access number CECT 9474.
    [4]
    4. Product for the control of the processionary of the pine (Thaumetopoea pityocampa) for its application for the degradation of the silk of its nests or pockets, characterized according to the first claim, because it is materialized in the strain Bacillus licheniformis RGA3 and deposited in the CECT with the access number CECT 9493.
    [5]
    5. Procedure for the control of the processionary of the pine (Thaumetopoea pityocampa) and the degradation of the silk of its nests or pockets, characterized according to the products of the claims 2 to 4, because it consists of the application of individual form, or in any other combination of said products, to processionary nests to achieve their disorganization and / or degradation, by spraying or any other alternative technique, favoring the efficacy of other potentially applied control methods.
    [6]
    6. Procedure for the control of the pine processionary (Thaumetopoea pityocampa) and the silk degradation of its nests or pockets, characterized according to the products of claims 2 to 4, because it consists of the application individually, or in any other combination of said products to liquid suspensions containing silk of procession to achieve its degradation and to obtain mixtures of amino acids or peptides of interest coming from it.
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