Isolation and pathogenicity of fungi associated to ambrosia borer ( Euplatypus segnis ) found injuring pecan ( Carya illinoensis ) wood

Euplatypus segnis is an insect pest of economic importance in pecan (Carya illinoensis) trees grown at Parras, General Cepeda and Torreón Coahuila, Mexico. The objectives in this studywere to identify the fungal strains associated to ambrosia borer body and diseased pecan wood and determine their pathogenicity. The results showed that the associated fungi to Euplatypus segnis and damaging the pecan wood were identified as: Helminthosporium sp., Aspergillus sp., Penicillium sp., Phoma sp., Ascochyta sp., Phaecylomices sp., Umbeliopsis sp., Torula sp., Fusarium solani, Alternaria alternata, Fusarum oxysporum, and Lasiodiplodia theobromae. The pathogenicity tests on healthy 3 year old pecan trees cv. western using Fusarium oxysporum, Fusarium solani, Alternaria alternata and Lasiodiplodia theobromae suspension conidia shown die back tree branches after 84 days inoculation. The insect in combination with the fungal invasion eventually cause the death of trees. Additionally, the insect contributes to the spread of fungi in pecan nut orchards.


INTRODUCTION
In Mexico, Euplatypus segnis also known as trunk and branch borer is distributed in the states of Coahuila, Durango, San Luis Potosi, Jalisco and Chihuahua [1][2][3].In the Coahuila Southwest region there are approximately 525 thousand pecan (Carya illinoensis) trees on produc-tion, and annually are harvested about 19,345 tons of nuts.However, in more that 20 percent of the tree plantations the presence of ambrosia borer Euplatypus segnis has been reported causing yield losses of up to 4% (773 tons of nuts) [4].In addition, E. segnis (Chapuis) (Coleoptera: Platypodinae), attacks wild trees such as: Bursera copallifera, Delonix regia, Ficus sp.Ficus lyrata, Ficus microcarpa, Ficus elastica and Ficus cotinifolia [5], fruit trees such as avocado, apple, peach, pomegranate, apricot, and mango, and some forestry trees like silver poplar, blackberry and tropical ash [6].There are reports on symbiotic associations between ambrosia insects and fungus, this relation help both of them to survive, per example Xyleborus ferrugineus and Lasiodiplodia theobromae in avocado (Persea americana Mill) [7], Xyleborus ferrugineus and Fusarium solani, Cephalosporium sp. and Graphium sp.[8], Ips typographus and Ophiostoma polonicum on Picea abies [9], Hypotenemus hampei and Fusarium solani on Coffea genus trees [10].Platypus quercivorus and Raffaelea quercivora on Quercus crispula and Q. serrata [11], Hypothenemus hampei and Penicillium brocae on Coffea arabiga [12], Platypus mutatus associated with a symbiotic fungus in commercial plantations of poplar [13].In this study, fungi associated to ambrosia borer isolated from diseased pecan wood were morphologically and molecularly identified and incidence of diseases induced by these fungal species was determined using pathogenicity tests.

Study Area
The sample collections of E. segnis specimens and pecan damaged tissues were obtained from Parras de la Fuente (25˚22'N 102˚11'W 1520 m), General Cepeda (25˚22'N 101˚28'W 1470 m) and Torreon (25˚42'N 103˚27'W 1120 m) all of these counties located in Coahuila, Mexico.Three orchards were sampled by locality and at each orchard were selected three 20-years old damaged trees cv.Western, the damaged trees were in the phase 3 (50% damaged leaf area, 25 to 50 holes entries and presence of sawdust at the stem base) [14].Wood and insects samples were placed in plastic bags and were properly coded and carried to the entomology and phytopathology laboratories for examination.

Fungal Isolates
The fungi were obtained from xylem, phloem and wood contained entry points and borer insects.The borer insects were sectioning into head, thorax and abdomen.Each section was disinfected used sodium hypochlorite (1.5%) by immersion for 3 min, after those insect sections were washed with sterile distilled water for 1 min and dried on sterile paper towels.Wood and insect sections were placed on Petri dishes containing potato dextrose agar (PDA: 20 g potato, 20 g of dextrose, 18 g agar and 1000 mL distilled water) as a culture medium, placing 4 pieces per plate.The Petri dishes were incubated and a continuous black light lamp 40 W to 25˚C ± 1˚C was using during the day.The isolates were purified by monospore cultures on water-agar (AA: 18 g agar in 1000 mL distilled water), and increased on PDA.The strains were spread on liquid medium (Pontecorbo) in order to obtain spores and mycelium production for pathogenicity test, and freeze drying and preservation.

Fungal Morphological Characterization
After 7 -10 days, the isolated and purified fungi from wood and insects were observed under a composed microscope and identified by morphology using taxonomic keys for ascomycetes and imperfect fungi, in this case characterization was based on characteristics of isolate reproductive structures using Barnnet and Hunter [15], Hanlin [16], Boot [17], Rotem [18], Neergaard [19], Sutton [20] keys for genus and Wei et al. [21] key for species.To identify Lasiodiplodia theobromae were used Punithalingam [22], Sutton [20] and Burgess et al. [23] keys.

Molecular Identification of Isolated Fungi
To confirm the morphological identification of those fungi colonies that showed the highest pathogenicity on pecan trees under field conditions.DNA was isolated from Fusarium oxysporum, Fusarium solani, Alternaria alternata and Lasiodiplodia theobromae fungi at the Molecular Biology laboratory, CIIDIR-IPN Oaxaca.The DNA was isolated using the methodology proposed by Ahrens and Seemüller [24].Amplification of internal transcribed spaces of ribosomal genes (rRNA) was performed by PCR using the primers ITS4 (TCC TCC GCT TAT TGA TAT GC) and ITS5 (GGA AGT AAA AGT CGT AAC AAG G) according to the methodology proposed by Ahrens and Seemüller [24] with minor modifications, PCR reaction was composed of sterile ultrapure water (13.22 μL), 10X TBE buffer (2.5 μL), MgCl 2 at 2.5 mM (2.08 μL), dNTPs at 0.2 mM (2 μL), primers ITS4 and ITS5 to 20 ρmol (2 μL of each), DNA polymerase (biogenic ® ) 1 U (0.2 μL) and 80 ng of DNA sample (1 uL).The amplified product was purified with Wizard kit (Promega ® ) and sequenced with the Genetic Analyzer ® Model 3100 (Applied Biosystems).The data base sequences with the highest value of similarity in were considered for comparison with the sequences obtained in this study.The ITS sequences obtained were analyzed using the software Lasergene ® 2001, V5 (DNASTAR ® , Inc.) and were aligned in the database of the National Gene Bank Center for Biotechnology Information (NCBI), by the BLAST program (Basic Local Alignment Search Tool) (http://www.ncbi.nlm.nih.gov/BLAST/).

Pathogenicity Tests
Inoculum was incremented in PDA culture medium which was autoclaved three times continues with an interval of 24 hours among sterilizations.Then were inoculated each of the twelve fungi isolated from pecan damaged wood and borer insect body: Phoma, Fusarium oxysporum, Ascochyta, Phaecylomices sp Lasiodiplodia theobromae, Alternaria alternata, Umbeliopsis sp, Torula sp, Fusarium solani, Helminthosporium sp, Aspergillus sp and Penicillium sp.The isolates were multiplied in the culture media described above, then were kept in an incubator at 27˚C with a photoperiod of 12:12 and a relative humidity of 50% ± 10%.Once sporulation was observed, the conidia from each isolate were harvested adding to each Petri dish 50 mL of a solution of Tween 80 (0.05%) and sterile distilled water, and removing the mold with a spatula.Subsequently, the mold solution was liquefied for 60 sec and placed in a beaker of 250 mL (stock suspension).Then, were made three successsive dilutions of 100 uL of the stock suspension in 900 uL of sterile distilled water and were labeled 10 -1 , 10 -2 and 10 -3 .Finally, spore concentration was determined using a Neubauer's camera and microscope, the dilution samples were observed in 40× to calculate spore concentration of the stock solution which was adjusted to a final concentration of 10 9 conidia/mL.
Pathogenicity tests were performed in vivo in twenty eight three years-old healthy pecan trees cv western (height (150 cm) and stem diameter (4 -5 cm)) established in a high technology greenhouse at a temperature of 24˚C ± 2˚C, at the Universidad Autonoma Agraria Antonio Narro located in Saltillo, Coahuila.The inoculation technique used in this step was that proposed by Kuroda [11], briefly; trees bark at a height of 100 cm was sterilize with sodium hypochlorite (1.5%), then washed with sterile distilled water and dried on using sterile absorbent paper.To each tree, two holes were made on the stem until xylem with a sterile 3-mm drill diameter.Twenty four trees were inoculated with 1 mL of the conidial suspension at 1 × 10 9 conidia•ml -1 of each one of the twelve identified fungi and two trees were inoculated with a mixed suspension of all fungi spores and two trees with sterile distilled water (control).The inoculations were added using a manual sterile pipette, each wound was covered with parafilm to prevent rapid drying and possible contamination by other microorganisms.According to this technique was favored the low density against mass inoculations in the inoculated tree to prevent wilting and rapid reactions to characterize tissues in a cytological way in response to the inoculated fungi activities.The evaluations were done every 7 days, watching symptoms and disease progression for 84 days after inoculation.Later, from the infected tissues of inoculated stems was isolated the fungus in pure culture and compared morphologically with the fungus inoculated to confirm Koch's postulates.

Statistical Analysis
The response variable was the presence of the fungal specie each case, which was evaluated in a quality way (presence and absence).For results analysis a categorical data analysis was performed.In this case Sx2 tables were used, where row and columns were nominally classified.Statistical analysis was performed using SAS (8.1 versions).

Disease Symptoms in Pecan Plantations under Field Conditions
In 20 years-old pecan trees were observed the characteristic symptoms of regressive dead and insect presence mainly in weakened and/or stressed (because age, water deficiency, injuries, fires, intense cold, snow or prolonged drought) trees.In trunk and branches were detected 2 mm-diameter entry holes and sawdust at the trees base, crystal reddish exudates that eventually became dark brown and pungent, after, taking off the bark, necrotic spots were observed with the typical form of diamond.On the other hand one cans heard a sound in trunks and branches; in addition one can see the sap out of the branches when phloem and xylem are piercing by the borer.Also the presence of adults, especially during morning and evening is detected, chlorosis is observed in the leaf area, wilting, premature abscission of stems and leaves, partial death of branches and downward total death of trees.

Morphological and Molecular Identification of Plant Pathogenic Fungi Associated with Plant Damage
The morphological characterization based on reproductive structures of the fungal isolates was performed using the taxonomic keys proposed by Barnnet and Hunter [15], Hanlin [16], Boot [17], Rotem [18], Neergaard [19], and Sutton [20] for genus and Wei et al. [21] keys were used for the specie identification.Lasiodiplodia theobromae was identified using the Punithalingam [22], Sutton [20] and Burgess et al. [23] keys.In Table 1 are shown the fungal species associated to die-back in pecan damaged wood.
The identification to genera and specie level by sequencing of the regions ITS1 and ITS2 of ribosomal genes (rRNA) was performed only to confirm identity of those fungal more common as pathogens.L. theobromae Table 1.Genus and species identified in samples of pecan with dieback symptoms and presence of ambrosia insects.

Fusarium oxysporum
Microscopically Fusarium oxysporum isolates showed abundant macroconidios, hyaline, oval, elliptical and occasionally kidney-shaped, with one or two septa, which measured 4 -5 × 2 -4 um, formed in false heads.The thin-walled hyaline macroconidia are just curved (canoe) or nearly straight, pointed at both ends, in the form of basal cell foot.Presented 3 -5 septa and measure 20 -59.5 × 2 -6 microns.It has also marked by abundant spherical chlamydospores, large, hyaline, with smooth or rough walls.Can be seen individually or in pairs at intervals along the hyphae or branched short lateral sections.

Alternaria alternata (fr,) Keissler
Alternaria alternata (fr) Keissler presents colony dark green to olive, conidia (dictyosporae) in chains on short conidiophores and branching.The strings appear in dense cultures and isolated as bundles of 1 to 9 transverse septa and several longitudinal or oblique.

Fungi Associated to Insects and Damaged Pecan Trees in the Coahuila Southwest Region
There were isolated 459 fungal strains from insects and diseased pecan wood on artificial culture medium (PDA), distributed as follows: In Parras county were isolated 165 strains, in General Cepeda county 161 strains, and in Torreón county 131 strains.The genera isolated were described according to their morphological structures based on microscopic observations and literature guides.
In the Table 2 are displayed the statistics for fungi by incidence.The Mantel-Haenszel Chi-Square (Qcs), is valid for these data since fungi is ordinally scaled, and the response is dichotomous; it indicates that there is a strong association between fungi and incidence (Qcs = 73.53)which is highly significant.By looking at the percentages in the Figure 1, we can see that fungi F. solani and F. oxysporum had the highest frequency of incidence.
In the Table 3 are displays the Mantel-Haenszel results for the stratified analysis where the strata are all combinations of county and tissue used (insect o wood) is Qcs MH = 73.4498,which is strongly significant, in this case some fungal species where observed only in some counties and a specific tissue (wood or insect) but no in others (Figures 2 and 3).The association of fungi and incidence controlling for counties is Qcs MH = 73.4878,which is highly significant.In this case some fungal species appeared only in samples taken in specific counties (Figures 3 and 4).On the other hand, the Mantel-Haenszel results for the stratified analysis where the strata are all combinations of tissue used (insect o wood) is Qcs MH = 73.5103,respectively, which is highly significant.In     this case some fungal species appeared only in samples taken in a specific tissue but not in the others (Figure 2).The significant association between incidence and fungal specie remains evident.Q GMH and Q SH are significant too in all the analyses.Eighty nine fungal strains were isolated from borers insect body (Euplatypus segnis) collected in pecan branches and trunks at Parras county, 35% of the isolates belongs to Fusarium solani, 15.2% to Fusarium oxysporum, 13.9% to Penicillium, 11.4% to Alternaria alternata, 7.6% to Phoma sp., 3.8% to Helminthosporium sp., Torula sp., Aspergillus sp., and 2.5% to Lasiodiplodia theobromae and Umbeliopsis sp., (Figure 2), also 76 fungal strains were isolated from diseased pecan wood, 38.7% belongs to Fusarium solani, 21% to Fusarium oxysporum, 19.4% to Phoma sp., 9.7% to Alternaria alternata, 8.1% to Penicillium sp., and 3.2% to Phaecylomices sp.(Figure 2).
From the samples collected in the Torreon county, sixty nine fungal stains were isolated from borer insect (E.segnis) body collected attacking pecan branches and trunks.These strain were distributed as follow: 38 4).

Pathogenicity of Fungi Strains Isolated from Pecan Trees with Dieback
At 14 days after inoculation were observed the first signs of disease in some trees (Table 4).In trees inoculated with Fusarium solani had discharge in the drilling at 21 and 28 days, chlorosis at 42 and 49 days, wilting at 63 and 70 days and dieback after 77 days after inoculation, the inoculated trees with Fusarium oxysporumshown exudates at the inoculation point in the two treatments at 14 and 21 days, and brown leaves at 35 and 42 days, chlorosis at 45 and 56 days, at 63 and 70 days caused wilting and dieback at 77 and 84 days after inoculation.The inoculated trees by Alternaria alternata showed chlorosis at 63 days, wilting to 70 days and dieback to 84 days after inoculation.The trees infected with Lasiodiplodia theobromae presented leaves chlorosis at 56 days, premature abscission at 70 days, defoliation at 77 days and dieback at 84 days, the other tree shoots inoculated only presented reddish tip at 77 and chlorosis at 84 days; Torula sp.Showed brown leaves at 77 days and defoliation at 84 days, the second treatment presented only brown leaves at 84 days, the two treatments behaved similarly.Phoma sp., caused wilting only 70 days to complete the evaluation at 84 days, the trees inoculated with Phaecylomices sp.presented chlorotic symptoms at the end of the evaluation, Ascochyta sp., caused brown leaves at 63 days, wilting from 77 to 84 days, in the second treatment was observed brown leaves at 70 days and defoliation at 84 days; Helminthosporium sp., caused chlorosis at 77 days and wilting at 84 days, the other treatment only showed a slight chlorosis; The treatments that received a mixture of spores showed the most damaged where trees were killed at 77 and 84 days in both treatments, Penicillium sp. and Aspergillus sp.Umbeliopsis sp. and the control treatment did not cause any outward symptoms.

Insect-Fungi Association
Euplatypus segnis is is an ambrosia specie with monogenic reproductive behavior [25], polyphagous in its degree of specificity with respect to the host and xylomicetophagus by feeding habit, consuming the conidia producing by  the fungi inoculated to the trees [26].Ulloa [27] mentions that borers have the habit of feeding on fungi introduced by them in the gallery system built into the wood.These fungi appear to be relatively non-specific and need basically moisted wood for their development.
Insect species that grow fungus in galleries are called beetles or ambrosia borers and they are dominant in tropical areas [28].Most of the buildings in the tunnel systems of ragweed borer (galleries) are in the vigorous host tissues, weakened or recently dead, though some species are specialize in colonize the bone marrow, large seeds, fruits and petiole leaves [26,29].The term ambrosia refers to the fungi that are cultivated by beetles in the gallery walls, which are feed exclusively for insects.
Beetles are necessarily dependent on fungi, from which acquired vitamins, amino acids, and essential sterols [30,31].The fungi are the main source of food for larvae and adults and are essential to complete insect life cycle [10].
In some insect tribes, only females perform tasks inside the culture, while males are short lived and little flight [32].After mating, females disperse to new host substrate, carrying fungi in specialized pockets called mycangia.Once inside, the founder females planted the fungi on the walls of tunnels, lay eggs, and serve the growing brood [32].In a way not understood, the female beetles can control fungal growth and degree of multispecies composition [30,[33][34][35].If the female dies, the culture is quickly contaminated by other fungi and bacte-ria, which ultimately leads to the brood death [32,36].The ambrosia fungi cultured by borers are not pure monocultures, also which consist of a mixture of fungi mycelium, yeasts, and bacteria [37,38].Norris [39] calls these complex multi-species mixtures.However, recent work has shown that a fungus always dominates in primary cultures of the insect [37,[40][41][42].In addition, beetles carry only the primary fungus in the mycangia (although sometimes secondary fungi are isolated from the mycangia), females tend to favor the primary fungal culture, which gives them a nutritional benefit [32,41,43,44], some auxiliary fungi also support beetle development [32].These observations imply the primary fungus as the primary crop, while fungi, yeasts and bacteria may be secondary "weeds" or with additional auxiliary roles to play in the cultures.The fungus growing transmission among generations of farmer's beetles is transmitted vertically from parent to offspring generations [38,43,45].
Reproductive females acquire original inoculum from their crops, they take with them in specialized pockets during dispersal flights and use this as seed inoculum for their new crops.Ambrosia borers are only associated with a particular kind of primary cultures within a specific geographic region [37,41].However, although most of the beetles are associated with a specific species of fungus in their primary area, ambrosia beetle species distantly related are sometimes associated with cultivating the same fungus, involving the exchange, direct or indirectly.The fungal exchange between and within species of beetles can occur when several females colonize the same tree and fungal associations contaminate adjacent galleries [41]).The primary fungi are primarily asexual [46,47], while less specific auxiliary fungi are often sexual [43].

Fungi Associated with Pecan in the Coahuila Southwestern Region
The identification of different fungal isolates from both wood and insect body, one may infer that diebackmay be associated with Fusarium since it is the fungal specie most frequently isolated from pecan tissue and insect body (Figure 1).
This association of Fusarium solani and Fusarium oxysporum as a primary agent on pecan tree is due to exudates or gummosis presented at 14 days, later inoculated trees showed marked chlorosis and red apices, finally trees declined to dead.Inside of stalks were initially observed brown areas and when these areas were cut transversely, staining was observed in the vascular system in ring forms, this situation was observed in leaf petiole.When the fungal pathogen completely invaded the conducting vessels caused widespread of wilt and tree death.From all the isolated fungal species, Fusarium solani and Fusarium oxysporum are highlighted in this study because they have a recognized importance as ambrosia fungus [30].Fusarium solani had the highest percentage of occurrence at Parras county, 35% in insects and 38.7% in diseased wood and in Torreon county, 38.5% in insects and 34.9% in diseased wood, by this reason it is considered as the primary ambrosia fungus, in contrast Fusarium oxysporum was the most frequent fungal specie at General Cepeda county, 45.9% in insects and 38.7% in diseased wood.As genera Fusarium are a ubiquitous necrotic parasite [48], and may tolerates pH changes [49], and host susceptibility to this fungus is directly influenced by temperature and osmotic pressure [50], Fusarium is widely distributed in soils and organic substrates, and it has been isolated from permafrost in the Arctic to Sahara sands, it presence is abundant in cultivated soils from temperate and tropical areas and it is the most frequently isolated fungi from plants by pathologists [17].Fusarium species are commonly associated with some group of insects like Scolytidae and Platypodidae families habit in wood [51].Regarding the association of Fusarium species and order Coleoptera insects in woods, Norris [32] mention Fusarium solani as a primary ambrosia fungus associated with species from Xyleborus genus, this report is consistent with the results obtained in this study, since 27.1% of our samples from the three different locations showed damage associated with insects of the Coleoptera order.Gil et al., [52] identified Corthylus sp.(Coleoptera: Scolytidae), causing damage to alder (Alnus acuminata) trees, which showed wood rot and death, in this case the inset was associated to some ambrosia fungi like Fusarium solani, Fusarium sp.Ceratocystis sp., Verticillium sp. and a yeast identified as Pichia sp.Bonello et al. [53] found that the ambrosia beetle Xylosandrus germanus associated with Fusarium lateritium and F. oxysporum, is related with vascular stained, cancers, wilting and dieback in black walnut (Juglans nigra).Tisserat et al. [54] found that the mortality of black walnut (Juglans nigra) in Colorado, USA, is the result of association of Pityophthorus juglandis (Coleoptera: Scolytidae) with Fusarium solani and Geosmithia sp. this fungus was latter isolated from trunk marginal cancer in the final stages of decline.F. solani has been mentioned as a primary symbiotic with the coffee berry borer (Hypothenemus hampei) [55,56].The immature stages of X. ferrugineus are unable to complete the development in absence of F. solani [34,[57][58][59].Ergosterol is essential for development and reproduction of X. ferrugineus [60,61].This sterol is absent in wood, but F. solani offer it in sufficient quantities [31].This fungus also provides to X. ferrugineus essential fatty acids and phospholipids [62].Norris [32] found that X. ferrugineus offspring was highly associated to the primary ambrosia fungus Fusarium solani, but with Cephalosporium sp.association was reduced by 50% and with Graphium sp. up to 70%.Pityophthorus juglandis is associated with F. solani in black walnut (Juglans nigra) trees in North America and South Africa; it is believed that trees are predisposed to cancer formation by stress factors such as optimum site conditions, improper pruning and adverse weather conditions [54], such as abandoned orchards and poor management.In our study, F. solani and F. oxysporum were consistently isolated from bone, xylem, phloem, cambium and galleries, as well as all Euplatypus segnis body parts.This observation and pathogenicity studies suggest an important role of F. oxysporum and F. solani in the pecan tree mortality of all ages in our region.
In the pathogenicity tests, Lasiodiplodia theobromae induced necrotic lesions approximately 10 cm long and 1.0 cm wide, that caused necrosis in the cortical tissue and deepened to the bone in the site of inoculation, at 56 days showed a slight chlorosis followed by early abscission, leaves with red tips, wilting and dieback tree at 84 days, the slow progression of the disease coincides with Milholl and [62] who mentioned that this type of fungus grow slowly and progress in living tissue host due to its saprophytic condition.Optionally, L. theobromae is a parasite that usually infects its host plants by penetrating through wounds and decaying tissue.Occurrence of this fungus is common in tropical and subtropical regions and in different ecological areas, where it has been identified as the cause of disease in approximately 280 species of vascular plants, including avocado, apple, mango, grapes, pine, rose, rubber, cotton, cocoa, coffee, sugar cane, peanuts, tobacco, etc. [63].Umezurike [64] mentions that Botryodiplodia theobromae has cellulolytic activity in Bombax buonopozense wood.The fungus attacks plant cells in a similar manner to soft rot fungi, use starch and other saccharides present in the initial wood substrates before degradation of cellulose and hemicellulose, but not degrade lignin.Rajput et al. [65] mention that decline and death of mango (Mangifera indica L.) trees in India, are related to the presence of Botryodiplodia theobromae, Alternaria alternata, Fusarium moniliforme, Cephalosporium sp., Chaetomium sp.Aspregillus ellipticus, A. niger, Penicillium sp., Curvularia lunata, Gloeosporium mangiferae, C. globosum, Daldinia sp associated to the attack of ambrosia beetles, termites, mechanical damage to trunk, branches and pruning.Rondon [7] established the relationship between the wood borer Xyleborus ferrugineus F. (Coleoptera: Scolytidae) and Botryodiplodia theobromae Pat attacking avocado trees in Venezuela, this author found that B. theobromae is the most frequently isolated fungi from the body of this wood borer insect.Flores et al. [66] detected the association of Scolytus sp. and Botryodiplodia theobromae fungus, which causes circular lesions to elongated oval up to 15 cm long, dark color that can be covered by large masses of spores in dry periods, causing the death of tips, and branches of Tectona grandis trees in Ecuador.Masood et al. [67] identified the association of Hypocryphalus mangiferae (Coleoptera: Scolytidae) with Lasiodiplodia theobromae, Phomopsis fimbriata and Ceratocystis sp. in the sudden death syndrome in Pakistan associated with symptoms of gummosis, dry rot and vascular discoloration.Tress inoculated with Alternaria alternata showed at 63 days marked chlorosis, wilting leaf area at 70 days and dieback at 84 days as external symptoms and necrotic areas, browning of tissues and a deep dehydration of inoculated area.The genus Alternaria contains cosmopolitan species found in a wide range of materials and products, can damage food and feed, producing biologically active compounds such as mycotoxins.As pathogens reduce crop yields or affect stored plants therefore requires a precise identification of the species because each has specific characteristics (preferences for growth, pathogenicity, and production of secondary metabolites) that predict the behavior of this fungus [68].Armengol et al. [69] mentioned that A. alternata is normally a saprophytic fungus, but in Spain some strains have been described as pathogenic on different citrus cultivars like Fortune, Nova, and Mineola.Negron et al. [70] described Alternaria as parasites of annual and bi-annual plants or saprophytes on organic substrates, however found that Alternaria alternata associated with Scolytus schevyrewi (Coleoptera: Scolytidae) and other fungi affect some species of elms (Ulmus spp) in Colorado and Utah, which showed necrotic areas, browning of xylem tissue, wilting of foliage and general death of elms, symptoms consistent with the disease progression in the pecan trees of this study inoculated with A. alternata in our pathogenicity tests.Reyes [71] found that 8% of fungal strains isolated from wood samples and insect remains of adult weevils (Coleoptera: Platypodidae) belong to Alternaria alternata and 7% to Phoma sp.
The fungus Paecilomyces sp. is a pathogen of wide host range and wide geographical distribution, which has been isolated from soil and insects of various orders such as Coleoptera, Homoptera and Collembola, Lepidoptera, Diptera, Homoptera, Hymenoptera and spiders [72].Paecilomyces farinosus may inhibit some strains of pathogenic fungi [73].Finally, Aspergillus, Paecilomyces, and Penicillium, have been isolated from insect gut Triatoma sp.[74], which has been found sporadically in pecan orchards at the Coahuila Southeast region, Mexico.

CONCLUSIONS
Twelve genera of fungi were isolated from the body of E. segnis and diseased pecan wood.These fungi were identified as Phoma sp., Fusarium sp., Ascochyta sp., Phaecylomices sp., Lasiodiplodia sp., Alternaria sp., Umbeliopsis sp., Torula sp., Helminthosporium sp., Aspergillus sp., and Penicillium sp., and inoculated into healthy pecan trees, only Fusarium oxysporum, Fusarium solani, Alternaria alternata and Lasiodiplodia theobromae were highly pathogenic causing dieback at 87 days of inoculation, they were characterized morphologically and molecularly to the specie level.
In Mexico, this is the first study that determines to Fusarium solani, Fusarium oxysporum, Alternaria alternata and Lasiodiplodia theobromae, as the causal agent of pecan (Carya illinoensis) dieback in association with Euplatypus segnis ambrosia borer.
Fusarium solani and F. oxysporum were the most prevalent fungi and isolated from insects and diseased wood at all studied locations.It has been suggested that some species of fungi associated with ambrosia borer are in a symbiotic relationship with the insect.Although, there may be a symbiotic relationship between fungi and insects, there is no conclusive evidence to show that this relationship is sufficient, due to the great diversity of fungi found in association with E. segnis.

Figure 1 .
Figure 1.Fungi species isolated from the Euplatypus segnis collected attacking pecan branches and trunks at three Coahuila counties.

Table 2 .
Statistics for fungi by incidence.

Table 3 .
Statistics for stratified by county and tissue used, stratified by county and stratified by tissue used.

Table 4 .
Development of symptoms and signs in pecan trees inoculated with fungal strains isolated from E. segnis and damaged pecan wood.