Paecilomyces niveus Stolk & Samson , 1971 ( Ascomycota : Thermoascaceae ) as a pathogen of Nasonovia ribisnigri ( Mosley , 1841 ) ( Hemiptera , Aphididae ) in Brazil

Nasonovia ribisnigri is a key pest of lettuce (Lactuca sativa L.) in Brazil that requires alternative control methods to synthetic pesticides. We report, for the first time, the occurrence of Paecilomyces niveus as an entomopathogen of the aphid Nasonovia ribisnigri in Pinhais, Paraná, Brazil. Samples of mummified aphids were collected from lettuce crops. The fungus P. niveus (PaePR) was isolated from the insect bodies and identified by macro and micromorphology. The species was confirmed by sequencing Internal Transcribed Spacer (ITS) rDNA. We obtained a sequence of 528 bp (accession number HQ441751), which aligned with Byssochlamys nivea strains (100% identities). In a bioassay, 120 h after inoculation of N. ribisnigri with pathogenic P. niveus had an average mortality of 74%. The presence of P. niveus as a natural pathogen of N. ribisnigri in Brazil suggests that it may be possible to employ P. niveus to minimize the use of chemical insecticides.

There is very little available information about the control of aphids using entomopathogenic fungi in Brazil.New fungal strains are important for N. ribisnigri control, and may lead to improvements in lettuce production.
We report, for the first time, the occurrence of P. niveus as entomopathogen of the aphid N. ribisnigri in Pinhais, Paraná, Brazil.

Collection, isolation, and identification of filamentous fungi
During the monitoring of the entomofauna in a commercial lettuce crop in Pinhais Country, Paraná, Brazil (25º 25' S and 49º 08' W, 930 m), dead specimens of N. ribisnigri with fungal mycelia growth on the body surface were found.The climate in the area was temperate according to Köppen as Cfb, (Peel et al., 2007), the average temperature was 21.2 ± 5 °C, and the relative humidity was 84 ± 10%.
Twenty mummified aphid specimens were collected during the fall.To promote fungal development and sporulation and to confirm that fungal infection was the cause of aphid death, specimens were placed on moist filter paper inside plastic Petri dishes, and then incubated for 7 days at 25 ± 1 °C under a 16:8 h light:dark photoperiod, and 60 ± 10% relative humidity (RH) Fungal mycelia grown on the surface of insect bodies and cultured for 7 days on potato dextrose agar medium (PDA) at 28 °C ± 1 °C.
Preliminary fungal identification was carried out by examining the macro and microscopic features of the colonies after slide culturing on PDA at 28 ± 1 °C (Hoog and Guarro, 2000).
Molecular identification of the fungus was performed by sequencing internal transcribed spacer (ITS) rDNA.Purified DNA was obtained as described in Gerrits van den Ende and Hoog (1999).The rDNA regions ITS1, 58 S, and ITS2 were sequenced using ITS5 and ITS4 primers (White Junior et al., 1990).Sequencing was performed in an automated sequencer (ABI 3700, Applied biosystems, Foster City, CA, USA).Sequences were edited and aligned using the Staden sequence analysis package v1.6.0 (Staden, 1996).Sequence analysis was performed using the sequence alignment software BLASTn, which was run against the NCBI's database (National Center for Biotechnology Information website).

Pathogenicity bioassays
Koch's postulates were used to determine the link between P. niveus and N. ribisnigri.In order to produce an inoculum, a suspension of the isolate was grown on PDA for 7 days at 28 °C ± 1 °C in Petri dishes and then incubated for an additional seven days at 25 °C ± 1 °C, a 16:8 h light:dark photoperiod under 70 ± 10% RH.The concentration of conidia in the filtrate was estimated using an improved Neubauer brightline hemocytometer (Reichart) under a Leitz Dialux 20 EB light microscope (400x).Suspensions were diluted to a final concentration of 1 × 10 8 conidia mL -1 .
A total of 100 third-instar N. ribisnigri nymphs were randomly assigned to a fungal treatment group and untreated control group.For each treatment, a 50-mm diameter leaf disc was cut out of a healthy lettuce plant.For the fungal treatment group, the leaf disc was dipped with 2 µL of conidial suspension using a micropipette.The leaf discs were then fed to aphids in Petri dishes containing filter paper moistened with sterile distilled water.Each dish contained 10 aphids.The aphids were then transferred to an environmentally controlled room (25 ± 1 °C, 16:8 h light: dark photoperiod with 70 ± 10% RH) and evaluated every 2 days for 10 days.Dead insects were collected and immersed in 70% ethanol for surface sterilization and were then transferred to individual Petri dishes containing moist filter paper and incubated for 7 days at 25 ± 1 °C under a 16:8 h light:dark photoperiod and 60 ± 10% RH.

Statistical analysis
Mortality data were corrected using the Abbott Formula (Abbott, 1925) and percentage values were arcsine transformed (√ (x/100)).Mean mortality data (fungus and water) were compared using the Scott-Knott test as implemented in Sisvar 5.3 software (Ferreira, 2010).Results were considered significant at the 0.05 level.

Results
Paecilomyces niveus (PaePR) was the only fungus recovered from N. ribisnigri collected from lettuce.After sequencing the PCR amplicon of the ITS regions of the fungal rDNA we obtain a sequence of 528 bp (Table 1).
Comparison of the obtained sequence to others in the database suggested that the isolate was from Byssochlamys nivea (FJ389938 with 100% of similarity), which is the teleomorphic phase of Paecilomyces niveus.The obtained sequence had 94-100% similarity to sequences from 21 strains of Paecilomyces sp., and these were used for phylogenetic analysis (Figure 1).The sequence was aligned and submitted to Genbank (accession number HQ441751).
A tree based on rDNA ITS sequencing was built using a neighbor-joining method and applying Jukes-Cantor correct distance model with 1000 bootstrap inferences, as implemented in Mega 4.0.2.Two major groups with high bootstrap values were obtained.The isolate PaePR was clustered with its teleomorph, B. nivea.
Pathogenicity assays showed significant difference between two treatments (F-value = 110.162p-value <0.001).N. ribisnigri showed a mean mortality of 74% 120 hours after inoculation with P. niveus.Control aphids remained asymptomatic and had a mortality of 12%.The pathogen was recovered from the insect body surface and the identitity of the pathogen was confirmed as P. niveus using morphological and molecular techniques (Figure 2).
According to Samson et al. ( 2009) the genus Byssochlamys is morphologically well defined and characterized by almost naked ascomata in which croziers and globose asci are formed with ellipsoidal ascospores.All Byssochlamys species have a Paecilomyces anamorph that belongs to the Paecilomyces sect.
The isolate was initially characterized as Paecilomyces sp. by macro-and micro-morphological analysis.ITS sequencing and phylogenetic analysis indicated that the isolate (PaePR) obtained in this study was clustered with its teleomorph, B. nivea.Samson et al. (2009)

Figure 1 .
Figure 1.Phylogenetic tree of Paecilomyces and Byssochlamys species based on ITS sequences.The tree was constructed using a neighbor-joining method, as implemented in MEGA 4.0.2.Bold branches indicate bootstrap values > 80 from 100 resampled datasets.The strain in bold, Paecilomyces niveus, was isolated as part of this study.The sequence for this strain is available in GenBank, accession No. HQ441751.

Table 1 .
verified by phylogenetic analyses that the genus Byssochlamys includes nine species, five of which form teleomorphs, i.e., B. fulva, B. lagunculariae, B. nivea, B. spectabilis and B. zollerniae, whereas four are asexual, namely P. brunneolus, P. divaricatus, P. formosus, and P. saturatus.This is the first report of P. niveus as a parasite of N. ribisnigri in Brazil.This study provides data for future research into the use of fungal isolates for the Fungal strains used for phylogenetic analysis of Paecilomyces niveus isolated from the aphid Nasonovia ribisnigri.