The Entomopathogenic Fungi Lecanicillium Sabanerum Sp. Nov. a Natural Control Agent of Parthenolecanium Sp

The entomopathogenic fungi Lecanicillium sabanerum sp. nov. a natural control agent of Parthenolecanium sp. (Hemiptera: Coccidae) in Bogotá, Colombia

Chiriví-Salomón, J.S.1, Sanjuan, T.1,2, Restrepo, S.1

1Laboratorio de Micologia y Fitopatología, Universidad de Los Andes, Colombia. 2Laboratorio de Taxonomía y Ecología de Hongos, Universidad de Antioquia, Colombia

Abstract

Parthenolecanium sp. is a scale insect that causes a candelabrum-like symptom in branches and a progressive terminal defoliation in trees of Ficus soatensis var bogotensis, an ornamental urban tree from Bogotá. Traditional pesticides were forbidden due to possible negative effects to human health, fauna and flora of the location. Natural epizootics of entomopathogenic fungi were observed in a residential zone of Bogota. Six districts were sampled to identify the entomopathogenic fungi. Environmental conditions were recorded from each sample site. Morphological characterization was developed for mycosed insects and isolates. Six nuclear loci were amplified: small subunit ribosomal RNA nu- rSSU, large subunit ribosomal RNA nu-rLSU, translation elongation factor 1-alpha- like (tef1), RNA polymerase I (B) subunit (Rpb1), RNA polymerase II (B) subunit (Rpb2) and internal transcribed spacer 1 5.8S ribosomal (nrITS). A phylogeny using Maximum Likelihood was performed for nrITS amplified sequences to identify the fungal specie, and a phylogenetic tree was constructed to place fungus in the Cordycipitaceae family. Lecanicillium sabanerum sp. nov. was proposed as a natural control agent of Parthenolecanium sp. and was placed in Cordycipitaceae family with bootstraps of 95 and 100 according to nrITS tree and phylogenetic tree, respectively. The influence of particulate matter concentration and other characteristics observed in the field on the incidence of entomopathogenic fungi in the city was discussed.

Key words: natural control, entomopathogenic fungi, Parthenolecanium, Ficus soatensis, Lecanicillium.

1. Introduction provides habitat to birds and offers shadow due to its size (Weisner, Ficus soatensis bogotensis var is a 2009). In addition, like others major ornamental tree in Bogotá that members of Moraceae family, this has the ability to tie to the land, tree has great ecological importance because its strong relation with wasps (Granara de Willink and Claps, 2003). that have in this kind of ficus a unique Female coccids secrete waxes that host (Cardona et al. 2007). Ficus tree protect them and their eggs by was chosen as emblematic tree of the filtrating toxic particles from their city in 1997 (Weisner, 2009), which environment (Foldi and Pearce, 1985; was replaced by Walnut Tree Gullan and Kosztarab, 1997). These (Juglans neotropica Diels) in 2002 protective compounds are produced (Concejo_de_Bogotá_Distrito_Capital, in glands of the epidermis of scale 2002). Ficus soatensis var bogotensis insect (Zhang et al., 2012). is in the arborization program of Parthenogenesis is another Bogota (Alcaldía Mayor de Bogotá characteristic that allocates an Distrito Capital, 2011) and its increasing in population density due population is about 100.000 the exclusive production of females. individuals. Additionally, the dissemination of the disease increases when males are In the last two years, Ficus population produced due to their capacity to fly has been affected by the scale insect (Gullan and Kosztarab, 1997; Nur, Parthenolecanium sp. (Hemiptera: 1971). Coccidae). These coccids cause a candelabrum-like symptom in branches and produce a massive The use of fungi as bioinsecticides defoliation until the tree dies (Fig. 1.). has been developed in the last 20 Coccids are important pathogens of years. Strains of species like higher plants due to their feeding Beauveria bassiana, Metarhizium habits and have been reported as anisoplae, Paecilomyces pest of ornamental plants (Peronti et fumosoroseus and Lecanicillium al., 2001; Van Driesche et al., 2010). lecanni have been used in biological Scale insects are sap sucking control with success (Vega and hemipterans that may inject toxins, Blackwell, 2004). All of them are transmit viruses and attract ants to grouped in Cordycipitaceae and plants (Kondo et al., 2008). Scale Hypocreaceae families, and they are population can exceed its density associated with larvae and pupae of when a tree is weak due to, for insects of the orders Hemiptera, example, its environmental conditions Lepidoptera and Coleoptera (Sung et (Fig. 1.). In this point, infestation al., 2007). Epizootics of becomes a problem, disease worsens entomopathogenic fungi have been and tree is even susceptible to other seen on Parthenolecanium population pests (Hanson and Miller, 1984). in the district of Barrios Unidos about Morphological features of these last year. Members of Hypocreaceae arthropods allow them surviving in family have been documented as unfavorable environmental conditions pathogens of scale insects (Chaverri Fig. 1. Candelabrum-like symptom in branches of Ficus tree, Parthenolecanium sp. (scale insects), epizootics of entomopathogenic fungi in scale insects. (Left to right). et al., 2008). These microorganisms annual precipitation of 1013 mm and could be an alternative to control a relative humidity of 72 %. Monthly these coccids in the Ficus population precipitation (PMPLL) were maximum (Fig. 1). The aim of this research is in months of march-may and october- the isolation and identification of december (Secretaría Distrital de entomopathogenic fungi specie that is Ambiente, 2011). a natural control agent of Parthenolecanium sp. in Bogotá. In 2.2. Collecting of addition, we aim to determine the entomopathogenic fungi environmental conditions that favor Scale insects with epizootics of behavior of the fungus as biological entomopathogenic fungi were control agent. collected from each Ficus sampled that presented symptoms of the disease described above. Samples 2. Materials and Methods were characterized according to their 2.1. Field collection size, color, height in the tree, position relative to sun light and environmental La Candelaria, Santa Fé, Teusaquillo, conditions. Also number of diseased Chapinero, Usaquén and Barrios trees and number of trees with Unidos were the districts selected for presence of entomopathogenic fungi sampling the entomopathogenic fungi were recorded per sample site. in Bogotá (Colombia). The weather Presence of epiphytic organisms was conditions of Bogotá were an average described per tree. Environmental temperature of 14 °C, an average conditions were determined according to maps of particulate matter (PM) (Slifkin and Cumbie, 1988). The with aerodynamic diameter < 10 μm morphology of internal mycelium, diameter (PM10), relative humidity and phialides and conidia were measured precipitations of the city (Álvarez et al., and compared with published 2008). Samples were taken to the description of different laboratory for morphological and entomopathogenic fungi. Description molecular characterization. Branches of the colony after 10 days of of approximately 10 cm with mycosed incubation in PDA, MEA and CZD coccid were collected for conservation was made in room laboratory of different disease stages in silica gel. conditions. Characteristics as color, size, consistency, elevation and 2.3. Isolation of folding were recorded. Color of fungi entomopathogenic fungi were described according to assigned Isolations were performed from color code patches (Watrud et al., collected insects with epizootics of 2006). Fungi were cultured in PDA at fungi. Cadavers were attached with environmental conditions to prepare vaseline to the lid of petri dishes to slides for both light microscope and induce expulsion of conidia or scanning electron microscope (SEM). ascospores on culture media (Nielsen SEM was performed by freezing et al., 2003). Potato dextrose agar circular cuts of 0.5 mm of diameter. (PDA) (Scharlau), malt extract agar Lengths and widths of phialides and (MEA) (Scharlau) and czapek’s dox conidia of each strain isolated were (CZD) (Scharlau) were the media measured. used for isolation and maintenance of 2.5. Molecular characterization strains. Samples of all strains were stored in glass flasks containing 4 mL Total genomic DNA was extracted of distilled water at environmental from collected fungi by the DNA conditions (Diogo et al., 2005). extraction protocol for basidiomycetes with some modifications (Gardes and Morphological 2.4. Bruns, 1993; Góes-Neto et al., 2005). characterization Total genomic DNA was isolated from Morphology of mycosed insects were cultures grown in SDY liquid media -1 -1 described according to body layers of (20 g L glucose, 20 g L yeast -1 scale insect (Zhang et al., 2012). extract, 10 g L casein protein) by Fungal features were determined using an adaptation of a DNA treating samples with lactophenol extraction protocol for basidiomycetes cotton blue (Thomas et al., 1991) and decay fungi (Jasalavich et al., 2000). Congo red solution (KOH 3%, Congo DNA concentration was read in red 10% and fuscidic acid 10%) Spectrophotometer ND-1000 (Nanodrop ©) and was adjusted to a Tabla 1. List of primers

final concentration of 100 ηg μL-1 with dNTPs 5 µM, and 0.2 µL of Taq DNA a refraction index (260/280) Polymerase (recombinant) 5 µ µL-1 approximately of 2.00. The nuclear (Fermentas)) was performed. PCR for gene region of internal transcribed SSU amplification was carried out spacer (nrITS) 1-5.8S-ITS2 of the using the following program: 95 °C for rDNA was amplified and sequenced 5 min (predenaturation), 35 cycles at to identify fungi and to define a 94 °C for 1 min, 55 °C for 1 min, relation with close groups. Five 72 °C for 1 min, with a final extension nuclear gene regions were amplified at 72 °C for 10 min. PCR for nrITS, and sequenced to place it in reported tef1, Rpb1 and Rpb2 amplification phylogeny for entomopathogenic was carried out using the fallowing fungi (Sung et al., 2007). These program: 94°C for 3 min regions were from small subunit (denaturation), 10 cycles at 94°C for (nrSSU) ribosomal RNA gene, large 30 s, 55 °C for 1 min, 72 °C for 2 min, subunit (nrLSU) ribosomal RNA gene, 35 cycles at 94 °C for 30 s, 50 °C for the elongation factor 1-α (tef1), RNA 1 min, 72 °C for 2 min, with a final polymerase I (B) subunit (Rpb1) and extension at 72 °C for 3 min. DNAs RNA polymerase II (B) subunit (Rpb2). amplified were sequenced by Sanger Each polymerase chain reaction sequencing method. Forward and (PCR) consisted of 2 µL of DNA reverse nucleotide sequences were extracted added to 23µL PCR mix assembled and cleaned with (0.5µL of each primers 100nmol Geneious 5.1.2v Program (Table 1), 2.5 µL of 10x Taq buffer + (Drummond et al., 2010). The

(NH4)2SO4 (Fermentas), 2.0 µL of amplified sequences were compared MgCl2 25mM (Fermentas), 0.5 µL with a DNA sequence database in the Tabla 2. Taxa used in Maximum Likelihood and edit with Fig Tree program tree for nrITS. (Rambaut).

3. Taxonomy

Lecanicillium sabanerum is proposed as new species, which is pathogen of Parthenolecanium sp. in Bogota. Morphological characterization of collected specimens and isolated strains combined with phylogenetic analyses of molecular data revealed GenBank using a similarity search that fungi do not match with L. lecanii, program of BLAST (Johnson et al., L. attenuatum and other related 2008) in NCBI portal. species. 2.6. Phylogenetic analysis Lecanicillium sabanerum J. S. Obtained and retrieved sequences Chiriví-Salomón, Sanjuan, T. & were aligned in MUSCLE software Restrepo, S., sp. nov. (Robert C., 2004) on CIPRES Colonies on PDA and MEA reaching Science Gateway portal (Miller et al., 1.3-1.8 cm diam. in 10 days at 2009). tef1 sequences were aligned in laboratory conditions, cottony with MAFFT server (Katoh et al., 2009) on exudate drops, yellowish white (PY), EMBL-EBI Web Services. Alignment with orange (O) to deep yellow (Y) editing was performed in BioEdit reverse. Colonies on CZA reaching Sequence Alignment Editor Program 1.4-1.7cm diam. In 10 days at (Hall, 1999). Ambiguously aligned laboratory conditions, cottony with low regions were excluded from relief and folding, yellowish white (PY), phylogenetic analysis and gaps were with orange (O) reverse (Fig. 2). treated as missing data. Maximum Phialides short, 13-19 x 1.0-2.0 x 0.5- Likelihood (ML) analysis was carried 1.0 μm, tapering to a narrow tip form, out for ITS nuclear gene region more or less erect conidiophores. sequences (Table 2). Phylogenetic Ellipsoidal conidia, 3.5-4.5 x 1.5-2.0 tree based in ML analysis was μm, formed in mucilaginous heads, constructed for concatenated 9.0-20 μm of diameter (Fig. 3).. On sequences for other genes (Table 3). the host cottony, pale yellow (PY), ML was performed using RAxML-VI- solitary epizootics or grouped in HPC v2.0 (Stamatakis, 2006; clusters, 0.5-1.5 mm diameter. Stamatakis et al., 2008) on CIPRES Phialides 13-19 μm x 1.0-2.0 μm x Science Gateway portal (Miller et al., 0.5-1.0 μm, tapering to a narrow tip 2009). Phylogenetic tree was read form. Ellipsoidal conidia 3.5-4.5 μm x

Fig. 2. Lecanicillium sabanerum colonies on PDA and CZA (Left to right).

Fig. 3. Microscopy of Lecanicillium sabanerum in lactophenol cotton blue. 1.5-2.0 μm. Three layers of infection above can perform another on scale insect. First layer: phialides morphotype: without folding. and conidia on epicuticle of scale insect, prosenchymal mycelia on exocuticle of scale insect. Second 4. Results layer: prosenchymal mycelia on endocuticle and formation zone of From 220 Ficus trees sampled, 190 scale insect. Third layer: crossing individuals were found with symptoms hyphae, surrounding mycelia of wax- or signs of the disease (candelabrum- secreting glands and fat bodies on like symptom, presence of insects in haemocoele of scale insect. (Fig. 4). trunk, branches or leafs or, instead Temperature optimum 16-20 °C. both, presence of protective waxes of coccids). Noteworthy those most Known distribution. 2.560 m.a.s., healthy trees had mosses and lichens Bogota, Cundinamarca, Colombia. in their surface, especially in lower Etimology. Lecani-, referring to the areas of tree. On the other hand, major component, -cillium, suffix entomopathogenic fungi were found taken from Verticillium, sabanerum, infecting coccids in 44 Ficus trees referring to location of the tree host of from diseased trees. Ficus in AK 7 CL Parthenolecanium sp., Ficus 28 address presented a natural soatensis var. bogotensis. control by fungi, but this zone was operated by botanical garden workers. Commentary. L. sabanerum presents Intervention consisted of introducing frequently a pale yellow (PY) color grafts of branches with and cottony mycelia on scale insects. entomopathogenic fungi. Fungi were But there are specimens with flat collected principally from dark areas white (FW) and grayish tan (LT) of the plant (lower leaf surface, lower colors, corresponding to young and little branches surface, and cracks in old infections, respectively. Also, branches and trunk) where sunlight is cottony texture is found in young limited. Entomopathogenic fungi were infections while tough texture in old found in an upper area of two trees ones. Fungi are collected most from AK 68 CL 49A, but area is below frequently below young branches of a pedestrian bridge and sunlight is the tree where sunlight is limited. limited almost most of the day. Fungi Epizootics of L. sabanerum is found were found in different stages of commonly in periods of highly humid infection with various sizes (maximum and rainfalls. Old colonies on PDA of 0.8 cm and minimum of 0.4 cm) and MEA performed a powdery and there are generally grouped in texture. Exudate drops are produced clusters. Dataset of environmental in first subcultures of fungi after conditions of sampled areas, the isolation. Colonies on CZA described number of diseased trees and the Fig. 4. Microscopy of epizootic of Lecanicillium sabanerum on scale insect in Congo red solution. number of trees with epizootics of family contained 4 taxa and showed a fungi infecting coccids were shown in bootstrap support of 100. Nectriaceae Table 4. This information was family was well supported with a overlapped and compared with bootstrap value of 99 and included 3 humidity, particulate matter and taxa. Hypocreaceae family, precipitation maps (Fig. 5-7). Simplicillium clade and Cordycipitaceae family were Sequences showed high similarity separated from Ophiocordycipitaceae Lecanicillium lecanii with (also named family and sister groups with a Verticillium lecanii ) (Sung et al., 2001; boostrap of 99. Similarly, Zare et al., 2000), Torrubiella sp. Cordycipitaceae family achieved a Cordyceps (Chaverri et al., 2005) and separation with a bootstrap of 100 confragosa (Spatafora et al., 2007), from both Hypocreaceae family and according to BLAST. The nrITS Simplicillium clade. Cordycipitaceae region of fungi did match L. lecanii family included 36 taxa. L. sabanerum with an average maximum identity of was placed in Cordyceps s.s. clade in 99%. ML tree for nrITS is well Cordycipitaceae family and supported with bootstrap values Cordyceps confragosa was set as above 95 (Fig. 8). Families in the sister specie of L. sabanerum (Fig. 9). multilocus phylogenetic tree were well supported and they were organized in L. sabanerum is placed as a new agreement with previous results specie of entomopathogenic fungi (Sung et al., 2007). Bionectriaceae because its morphological

Fig. 5. Humidity map and collections sites. Parthenolecanium sp. absence (green circles), Parthenolecanium sp. presence (pink circles), Lecanicillium sabanerum presence (blue circles), subhumid zone (dark purple), semidry zone (orange), dry zone (purple), humid zone (cyano).

Fig. 6. PM10 map and collections sites. Parthenolecanium sp. absence (green circles), Parthenolecanium sp. presence (pink circles), Lecanicillium sabanerum presence (red circles), 40 - 50 μg/m3 (dark red), 50 – 60 μg/m3 (red), 60 – 70 μg/m3 (orange), 70 – 80 μg/m3 (purple), 80 – 90 μg/m3 (dark ochre), 90 – 100 μg/m3 (ochre).

Fig. 7. Precipitations map and collections sites. Parthenolecanium sp. absence (green circles), Parthenolecanium sp. presence (pink circles), Lecanicillium sabanerum presence (blue circles), 700 – 800 (dark orange), 800 – 900 (orange), 900 – 1000 (ochre), 1000 – 1100 (green faint), 1100 – 1200 (green), 1200 – 1300 (dark green). characteristics and phylogenetic L. sabanerum performed several position. The SEM showed mycelium morphotypes according to age and and conidiophores with phialides culture media. On the other hand, L. reported above and conidia organized lecanii teleomorph (C. confragosa) in mucilaginous heads (Fig. 10). L. was phylogenetic compared with L. sabanerum was compared with L. sabanerum and a separation between lecanii because its highly similarity these species was shown. reported in BLAST. These two species differ in several microscopic 5. Discussion and cultural characteristics. Phialides L. lecanii is an entomopathogenic of L. sabanerum were longer and fungi that is reported as anamorph of thicker than L. lecanii (11-20(-30) μm Torrubiella genus in phylogenetic x 1.3-1.8 μm). Ellipsoidal conidia of L. analyses for nrITS sequences (Gams sabanerum were considerably larger et al., 2000). Nevertheless, recent than L. lecanii (2.5-3.5(-4.2) μm x 1- rigorous phylogenetic analyses 1.5 μm). L. sabanerum colonies were connected L. lecanii with the smaller than L. lecanii (1.5-2.0 cm), teleomorph of Cordyceps confragosa also texture of both fungi was similar, (Sung et al., 2007). On the other hand,

Fig. 10. Scanning Electron Microscopy (SEM) for Lecanicillium sabanerum the nrITS tree presented in this study a bootstrap of 100. Their short length indicated that the entomopathogenic and established relations in nrITS tree fungus characterized belong to indicates that collected and isolated singular specie, and it is separated fungi are grouped in singular specie. from other hemipteran pathogens. C. Otherwise, C. confragosa and L. confragosa was considered in our sabanerum were grouped in a clade analysis, and our fungi resulted with a phylogenetic difference of 100 distinct from this specie with a of bootstrap. As stated above, C. bootstrap of 95. L. attenuatum, confragosa and L. sabanerum Cordyceps tuberculta and represent different species. It is Akanthomyces pistilariformis were notorious that these fungi were different with a bootstrap of 95 and closely related, which fits with the their hosts are lepidopteran (Dennis, obtained high similarity with L. lecanii 1995; Sung et al. 2007; Zare and in BLAST. Gams, 2001), a very dissimilar insect order of Hemiptera. In agreement with Preliminary morphological morphological analysis discussed identifications showed that these fungi above, Lecanicillium sabanerum is could belong to the Lecanicillium Cordyceps proposed as a new species and is a genus, anamorph of s.s., natural control agent of which has been described as Parthenolecanium sp. Multilocus pathogens of some spiders and phylogenetic tree showed that L. members of Hemiptera order (Evans sabanerum is placed in Cordyceps s.s. and Hywel-Jones, 1997). Species of clade in Cordycipiticeae family, this genus have been used as according with the proposed biological control agents, like Lecanicillium attenuatum phylogenetic tree of Sung and in green collaborators (Sung et al., 2007). In peach aphids (Hemiptera: this multilocus analysis, L. sabanerum Aphidoideae) (Kim et al., 2008) and Lecanicillium lecanii individuals resulted in two clades with in brown soft scales (Hemiptera: Coccidae) (Liu et proving a similarity with L. lecanii in al., 2011), respectively. Coincidentally, conidia arrangement (Fig. 6). isolates of entomopathogenic fungi of Parthenolecanium sp. showed The presence of exudate drops could morphological characteristics that be related to C:N ratio of culture placed it in Lecanicillium genus. This media (Hutwimmer et al., 2010) but it genus is recognized by its colony has been suggested an enzymatic characteristics on PDA and MEA. activity with ecological role by fungi, L. lecanii Additionally, morphology and according to studies with arrangement of its conidiophores, (Rocha-Pino et al., 2011), Metharhyzium anisopliae phialides and conidia are important (Hutwimmer features to consider in this genus. et al., 2010) and Ophiocordyceps Characters of isolated fungi are sinensis (Kuo et al., 2005), among comparable with L. lecanii (also other species. L. lecanii is induced to commercially named Verticillium produce hydrophobins and chitinases lecanii), L. longisporum and L. in submerged culture and solid- nodulosum. Nevertheless, substrate culture (Rocha-Pino et al., morphology of isolated fungi present 2011). It has been shown that M. more similarities with L. lecanii than anisopliae secretes droplets of the other Lecanicillium spp. i.e. exudates that contain dextruxins, Diameter of growth for isolates is enzymes with insecticidal properties within growing range of L. lecanii at (Hutwimmer et al., 2010). O. sinensis, 20°C, colonies aspect are equal a Lepidoptera larvae (excluding that color reverse of entomopathogenic, exudates isolates in some cases changing to compounds with anti-tumour and anti- orange), phialides are short and bacterial activity, likely for competition present a tapering to tips, the with other organisms (Kuo et al., arrangement of ellipsoidal conidia is 2005). In accordance with these in mucilaginous heads at tips of the studies and the wide range of phialides (Fig. 5 and Fig. 7). On the metabolites that these fungi can other hand, the main character that produce, a chemical analysis of L. sabanerum differentiates these fungi is conidia exudates from sp. nov. size, because conidia of isolated fungi is recommended to consider for future are bigger than L. lecanii conidia works. Moreover, there is reported Lecanicillium (Zare and Gams, 2001). SEM that fungi infect scale photographs showed the same insects by penetrating the integument characteristics that features observed with mechanical force and secreting in light microscopy. Conidia organized extracellular enzymes (Liu et al., in mucilaginous heads were clearly 2011). noted at the tips of the phialides The emergence of these fungi morphology and reproduction of some seemed to be related with La Niña fungi can be affected by air pollutants phenomenon, a period of highly (Estrabou et al., 2004). Although PM10 humidity of weather. But there is no concentration in the district of relation between both humidity and Usaquén is low, the absent of natural precipitations with the disease of control evidence could be explain due Ficus or presence of epizootics of L. dissemination of fungi. Studies sabanerum sp. nov. (Table 4, Fig. 5 indicate that fungi from the genus and Fig. 7). Probably moisture Lecanicillium notoriously increase its condition inside trees is the persistence when is dispersed by determinant factor of incidence of thirds agents (Down et al., 2009). entomopathogenic fungi. Water is Moreover, cleaning routes of Bogotá retained in branches and leafs are made by different cleaning creating a moisture microhabitat, due companies per district. Pruning is to precipitations and humidity from La realized and cut branches are Niña phenomenon. On the other hand, transported by trucks. Vegetal presence of epiphytic organism and material and debris are incinerated particulate material concentration (pers. com. Vargas, G. from Unidad seem to be important factors to Administrativa Especial de Servicios consider. First, coccids were absent Públicos, 2012). Transportation may when moss and/or lichen covered the not be suitable because it resembles cortex of trees, because they may helping to the dissemination of generate a physical barrier by limiting coccids. Additionally, proliferation of the space where scale insects feed. fungi seems to be affected by pruning. Second, mycosed coccids were only observed in areas where concentration of particulate material 6. Conclusions was below 70-80 μg m-3, excluding collected material from AK 7 CL 28 Morphological characteristics of where Botanical Garden workers collected fungi place them in the intervened with grafts. The PM Lecanicillium genus, reported impacts in the health of plants by pathogens of hemipteran insects. injuring the leaf surface, decreasing Fungi belong to a single species, with the photosynthetic rate and causing a bootstrap that separate them from uptake deficiencies in the rhizosfere brother species (Akanthomyces (Prajapati, 2012). In this sense, aculeatus, Cordyceps tuberculta, fluctuations caused by PM could be Lecanicillium attenuatum, Cordyceps affect the physical-chemical condition confragosa, Isaria farinosa and and microbial communities of soil that Cordyceps cocciodioperitheciata). help to plant in disease suppression Multilocus phylogenetic tree indicate (van Bruggen et al., 2006). Also that the fungus is placed in Cordyceps s.s. clade analysis of produced exudates is (Cordycipitaceae family). recommended to determine the Lecanicillium sabanerum sp. nov. is metabolic content, which may be proposed as natural control agent of advantageous for the industry. Parthenolecanium sp. and it has a great potential as biological control agent. 7. Acknowledgements This work was supported by PM seems to be a determinant factor Secretaria Distrital de Medio in L. sabanerum sp. nov. incidence. Ambiente, especially by Germán Although both precipitations and Tovar, forestall engineer and director humidity are not definitive factors, of forestry department. Sergio Daniel both shadow and humidity can Rojas Jérez, Nancy Salomón de generate propitious microhabitats for Chiriví and José Luis Chiriví Mahecha fungal growing and eventuality control collaborated with collecting of of scale insects on a tree. Presence samples. Ricardo Antonio Rambal of moss and/or lichen likely may avoid Fattori made illustrations for Fig. 5, those scale insects to feed from Ficus Fig. 6 and Fig. 7, and vectorize Tree. Morphology and physiology of illustrations for Fig. 3 and Fig. 4. coccids, and cultural practices ease their dissemination on Ficus population. Cultural practices seem to 8. References be affecting the L. sabanerum proliferation too. Alcaldía Mayor de Bogotá Distrito Capital, 2011. Programa de An epidemiological study of diseased arborización de Bogotá. caused by scale insects is Documento de arborización recommended for setting a correct urbana, Bogotá D.C. cleaning program in Bogota city. A Álvarez, G.D., Tovar, G., Bocanegra, suitable cleaning program will avoid a F., Chaparro, J.A., Caicedo, G., rapid dissemination of disease and 2008. Manual de Silvicultura permit a better control with urbana para Bogotá. Alcaldía entomopathogenic fungi. This Mayor de Bogotá, Bogotá D.C. cleaning program could include a Cardona, W., Chacón de Ulloa, P., pruning that provides microhabitats of Kattan, G., 2007. Avispas no shadow and humidity to maintaining polinizadoras asociadas a recycling and persistence of L. Ficus andicola (Moraceae) en sabanerum. On the other hand, la Cordillera Central de pathogenicity test of isolated strains is Colombia. Revista Colombiana important to guaranteeing a good de Entomología 33, 165-170. biological control. Finally, a chemical Castlebury, L. A., Rossman, A. Y., Brasileiros de Dermatologia 80, Sung, G. H., Hyten, A. S., 591-594. Spatafora, J. W., 2004. Down, R.E., Cuthbertson, A.G.S., Multigene phylogeny reveals Mathers, J.J., Walters, K.F.A., new lineage for Stachybotrys 2009. Dissemination of the chartarum, the indoor air entomopathogenic fungi, fungus. Mycological Research Lecanicillium longisporum and 108, 864-872. L. muscarium, by the predatory Concejo de Bogotá Distrito Capital, bug, Orius laevigatus, to 2002. Acuerdo 69 de 2002 provide concurrent control of Concejo de Bogotá Distrito Myzus persicae, Frankliniella Capital. In: Capital, occidentalis and Bemisia S.G.d.l.A.M.d.B.D., (Ed.), tabaci. Biological Control 50, Registro Distrital 2715 del 13 172–178. de septiembre de 2002, Drummond, A.J., Ashton, B., Buxton, Bogotá D. C. S., Cheung, M., Cooper, A., Chaverri, P., Bischoff, J.F., Evans, Duran, C., Field, M., Heled, J., H.C., Hodge, K.T., 2005. Kearse, M., Markowitz, S., Moir, Regiocrella, a new R., Stones-Havas, S., Sturrock, entomopathogenic genus with S., Thierer, T., Wilson, A., a pycnidial anamorph and its 2010. Geneious v5.1.2. phylogenetic placement in the Estrabou, C., Stiefkens, L., Hadid, M., Clavicipitaceae. Mycologia 97, Rodríguez, J.M., Pérez, A., 1225-1237. 2004. Effects of air pollutants Chaverri, P., Liu, M., Hodge, K.T., on morphology and 2008. A monograph of the reproduction in four lichen entomopathogenic genera species in Córdoba, Argentina. Hypocrella, Molleriella, and Ecología en Bolivia 39, 33-45. Samuelsia gen. nov Evans, H.C., Hywel-Jones, N.L., 1997. (Ascomycota, Hypocreales, Entomopathogenic fungi. In: Clavicipitaceae), and their Ben-Dov, Y., Hodgson, C.J. , aschersonia-like anamorphs in (Ed.), Soft Scale Insects: Their the Neotropics. Studies in Biology, Natural Enemies and Mycology, 1-66. Control. El Sevier, Amsterdam, Dennis, R.W.G., 1995. Fungi of South New York, pp. 15-16. East England. Royal Botanic Foldi, I., Pearce, M.J., 1985. Fine Gardens, Kew (RBG (K)). Structure of Wax Glands, Wax Diogo, H.C., Sarpieri, A., Pires, M.C., Morphology and Function in 2005. Fungi preservation in the Female Scale Insect, distilled water. Anais Pulvinaria regalis canard. (Hemiptera: Coccidae). Int. J. Insect MorphoL & Embryol 14, Perspective. Journal of 259-271. Arboriculture 10, 259-264. Gams, W., Culham, A., Zare, R., 2000. Hsieh, L.S., Tzean, S.S., Wu, W.J., A revision of Verticillium sect. 1997. The genus Prostrata. I. Phylogenetic Akanthomyces on spiders from studies using ITS sequences. Taiwan. Mycologia 89, 319-324. Nova hedwigia 71, 465-480. Hutwimmer, S., Wang, H., Strasser, Gardes, M., Bruns, T.D., 1993. ITS H., Burgstaller, W., 2010. primers with enhanced Formation of exudate droplets specificity for basidiomycetes - by Metarhizium anisopliae and application to the identification the presence of destruxins. of mycorrhizae and rusts. Mycologia 102, 1–10. Molecular Ecology 2, 113-118. Jasalavich, C.A., Ostrofsky, A., Góes-Neto, A., Loguercio-Leite, C., Jellison, J., 2000. Detection Guerrero, R.T., 2005. DNA and identification of decay extraction from frozen fungi in spruce wood by fieldcollected and dehydrated restriction fragment length herbarium fungal basidiomata: polymorphism analysis of performance of SDS and amplified genes encoding CTAB-based methods. rRNA. Appl Environ Microbiol Biotemas 18, 19-32. 66, 4725-4734. Granara de Willink, M.C., Claps, L.E., Johnson, M., Zaretskaya, I., Raytselis, 2003. Cochinillas (Hemiptera: Y., Merezhuk, Y., McGinnis, S., Coccoidea) Presentes en Madden, T.L., 2008. NCBI Plantas Ornamentales de la BLAST: a better web interface. Argentina. Neotropical Nucleic Acids Research 36, Entomology 32, 625-637. W5–W9. Gullan, P.J., Kosztarab, M., 1997. Katoh, K., Asimenos, G., Toh, H., Adaptations in Scale Insects. 2009. Multiple Alignment of Annual Review of Entomology DNA Sequences with MAFFT. 42, 23–50. Bioinformatics for DNA Hall, T.A., 1999. BioEdit: a user- Sequence Analysis 537, 39-64. friendly biological sequence Kim, Yeon, H., Lee, H., Kim, Y., Kim, alignment editor and analysis I., 2008. Laboratory and Field program fro Windows 95/98/NT. Evaluations of Nucleic Acids Symposium Entomopathogenic Series 41, 95-98. Lecanicillium attenuatum CNU- Hanson, P.E., Miller, J.C., 1984. 23 for Control of Green Peach Scale Iinsects on Ornamental Aphid (Myzus persicae). Plants: A Biological Control Journal of Microbiology and Biotechnology 18, 1915-1918. Kondo, T., Gullan, P.J., Williams, D.J., Entomophthorales. Biological 2008. Coccidology. The study Control 28, 92-100. of scale insects (Hemiptera: Nur, U., 1971. Parthenogenesis in Sternorrhyncha: Coccoidea). Coccids (Homoptera). Revista Corpica - Ciencia y American Zoologist 11, 301- Tecnología Agropecuaria 9, 308. 55-61. Peronti, A.L.B.G., Millery, D.R., Kuo, C., Chen, C., Luo, Y., Huang, Sousa-Silvaz, C.R., 2001. R.Y., Chuang, W., Sheu, C., Scale Insects (Hemiptera: Lin, Y., 2005. Cordyceps Coccoidea) of ornamental sinensis mycelium protects plants from Sao Carlos, Sao mice from group A Paulo, Brazil. INSECTA streptococcal infection. Journal MUNDI 15, 246-255. of Medical Microbiology 54, Prajapati, S.K., 2012. Ecological 795–802. effect of airborne particulate Liu, W., Xie, Y., Xue, J., Zhang, Y., matter on plants. Zhang, X., 2011. Ultrastructural Environmental Skeptics and and cytochemical Critics 1, 12-22 characterization of brown soft Rambaut, A., FigTree v1.3.1. scale Coccus hesperidum Robert C., E., 2004. MUSCLE: (Hemiptera: Coccidae) infected multiple sequence alignment by the Lecanicillium lecanii with high accuracy and high (Ascomycota: Hypocreales). throughput. Nucleic Acids Micron 42, 71-79. Research 32, 1792-1797. Liu,Y., Whelen, S., Hall, B., 1999. Rocha-Pino, Z., Vigueras, G., Shirai, Phylogenetic relationships K., 2011. Production and among ascomycetes: evidence activities of chitinases and from an RNA polymerase II hydrophobins from subunit. Molecular Biology Lecanicillium lecanii. Evolution 16, 1799-1808. Bioprocess Biosyst Eng 34, Miller, M.A., Holder, M.T., Vos, R., 681-686. Midford, P.E., Liebowitz, T., Secretaría Distrital de Ambiente, 2011. Chan, L., Hoover, P., Warnow, Observatorio Ambiental de T., 2009. The CIPRES Portals. Bogotá. Bogotá D.C. CIPRES. Slifkin, M., Cumbie, R., 1988. Congo Nielsen, C., Hajek, A.E., Humber, red as a fluorochrome for the R.A., Bresciani, J., Eilenberg, rapid detection of fungi. J Clin J., 2003. Soil as an Microbiol 26, 827-830. environment for winter survival Spatafora, J.W., Sung, G.H., Sung, of aphid-pathogenic J.M., Hywel-Jones, N.L., White, J.F.J., 2007. Phylogenetic evidence for an animal Microbiology and Infectious pathogen origin of ergot and Disease 14, 219-224. the grass endophytes. van Bruggen, A.H.C., Semenov, A.M., Molecular Ecology 16, 1701- van Diepeningen, A.D., de Vos, 1711. O.J., Blok, W.J., 2006. Relation Stamatakis, A., 2006. RAxML-VI- between soil health, wave-like HPC: Maximum Likelihood- fluctuations in microbial based Phylogenetic Analyses populations, and soil-borne with Thousands of Taxa and plant disease management. Mixed Models. Bioinformatics European Journal of Plant 22, 2688-2690. Pathology 115, 105–122. Stamatakis, A., Hoover, P., Van Driesche, R.G., Carruthers, R.I., Rougemont, J., 2008. A Fast Center, T., Hoddle, M.S., Bootstrapping Algorithm for the Hough-Goldstein, J., Morin, L., RAxML Web-Servers. Smith, L., Wagner, D.L., Systematic Biology 57, 758- Blossey, B., Brancatini, V., 771. Casagrande, R., Causton, C.E., Sung, G.H., Hywel-Jones, N.L., Sung, Coetzee, J.A., Cuda, J., Ding, J.M., Luangsa-Ard, J.J., J., Fowler, S.V., Frank, J.H., Shrestha, B., Spatafora, J.W., Fuester, R., Goolsby, J., 2007. Phylogenetic Grodowitz, M., Heard, T.A., Hill, classification of Cordyceps and M.P., Hoffmann, J.H., Huber, J., the clavicipitaceous fungi. Julien, M., Kairo, M.T.K., Kenis, Studies in Mycology, 5-59. M., Mason, P., Medal, J., Sung, G.H., Spatafora, J.W., Zare, R., Messing, R., Miller, R., Moore, Hodge, K.T., Gams, W., 2001. A., Neuenschwander, P., A revision of Verticillium sect. Newman, R., Norambuena, H., Prostrata. II. Phylogenetic Palmer, W.A., Pemberton, R., analyses of SSU and LSU Panduro, A.P., Pratt, P.D., nuclear rDNA sequences from Rayamajhi, M., Salom, S., anamorphs and teleomorphs of Sands, D., Schooler, S., the Clavicipitaceae. Nova Schwarzlander, M., Sheppard, hedwigia 72, 311-328. A., Shaw, R., Tipping, P.W., Thomas, P.A., Kuriakose, T., van Klinken, R.D., 2010. Kirupashanker, M.P., Classical biological control for Maharajan, V.S., 1991. Use of the protection of natural Lactophenol Cotton Blue ecosystems. Biological Control Mounts of Corneal Scrapings 54, S2-S33. as an Aid to the Diagnosis of Vega, F.E., Blackwell, M., 2004. Mycotic Keratitis. Diagnostic Insect-Fungal Associations: Ecology and Evolution. Oxford University Press, Inc., New III. Generic classification. Nova York. hedwigia 72, 329-337. Vilgalys, R., Sun, B. L., 1994. Ancient Zare, R., Gams, W., Culham, A., 2000. and recent patterns of A revision of Verticillium sect. geographic speciation in the Prostrata. I. Phylogenetic oyster mushroom Pleurotus studies using ITS sequences. revealed by phylogenetic Nova hedwigia 71, 465-480. analysys of ribosomal DNA Zhang, Y., Xie, Y., Xue, J., Fu, X., Liu, sequences. PNAS 91, 4599- W., 2012. The structure of 4603. integument and wax glands of Watrud, L.S., Martin, K., Donegan, Phenacoccus fraxinus K.K., Stone, J.K., Coleman, (Hemiptera: Coccoidea: C.G., 2006. Comparison of Pseudococcidae). Zoological taxonomic, colony morphotype Research 33, 13−17. and PCR-RFLP methods to characterize microfungal diversity. Mycologia 98, 384- 392. Weisner, D., 2009. Manual verde del caucho sabanero. Manual verde, Bogotá. White, T. J., Bruns, T. D., Lee, S., Taylor, J., 1990, Amplification

and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols, a guide to methods and applications. Academic Press,

San Diego, 315-222. Zare, R., Gams, W., 2001. A revision of Verticillium sect. Prostrata.