Naturalis® is a bioinsecticide based on living conidiospores of the naturally occuring

Beauveria bassiana strain ATCC 74040. The entomopathogenic fungus acts primarily by

contact: once attached to the insect’s cuticle, the conidiospores germinate producing

penetration hyphae, which enter and proliferate inside the insect’s body. The fungus

invades and feeds on its host, causing its death due to dehydration and/or depletion of

nutrients. Several years of laboratory, semi-field and field studies showed that also Tephritid

flies (Ceratitis capitata, Rhagoletis cerasi, Bactrocera oleae) are susceptible to infection by

B. bassiana strain ATCC 74040. The results of efficacy trials conducted in 2004-05 are

reported. Naturalis was tested both alone and in an integrated pest management strategy.

The product showed high efficacy in controlling R. cerasi, comparable to or higher than

that of the chemical reference treatment. The B. bassiana-based product Naturalis can thus

be considered an efficient tool for the control of the cherry fruit fly

Benuzzi, M.

Fiorentini, F.

Franceschini, S.

Ladurner, E.

English

Organic Eprints

93Beauveria bassiana strain ATCC 74040 (Naturalis®), a valuable tool for the control of the cherry fruit fly (Rhagoletis cerasi)E. Ladurner 1, M. Benuzzi 2, F. Fiorentini 3, and S. Franceschini 4AbstractNaturalis®  is  a  bioinsecticide  based  on  living  conidiospores  of  the  naturally  occuring Beauveria bassiana strain ATCC 74040. The entomopathogenic fungus acts primarily by contact:  once  attached  to  the  insect’s  cuticle,  the  conidiospores  germinate  producing penetration  hyphae,  which  enter  and  proliferate  inside  the  insect’s  body.  The  fungus invades and feeds on its host, causing its death due to dehydration and/or depletion of nutrients. Several years of laboratory, semi-field and field studies showed that also Tephritid flies (Ceratitis capitata, Rhagoletis cerasi, Bactrocera oleae) are susceptible to infection by B. bassiana strain ATCC 74040. The results of efficacy trials conducted in 2004-05 are reported. Naturalis was tested both alone and in an integrated pest management strategy. The product showed high efficacy in controlling R. cerasi, comparable to or higher than that of the chemical reference treatment. The B. bassiana-based product Naturalis can thus be considered an efficient tool for the control of the cherry fruit fly. Keywords:  Beauveria  bassiana, Rhagoletis  cerasi,  cherry,  organic  farming,  integrated pest management IntroductionBeauveria  bassiana  (Balsamo)  Vuillemin  (Deuteromycetes,  Moniliales)  is  an entomopathogenic fungus, recognized in 1835 by Agostino Bassi as the causal agent of the white muscardine disease of the silk worm. It can affect a wide range of arthropod pests, such as coleopterans, mites, and hemipterans, and all their developmental stages, but different B. bassiana strains differ in their host range (Knauf, 1992; Lacey et al., 1999; Mayoral et al., 2006; Talaei-Hassanloui et al., 2007).Naturalis® is a bioinsecticide based on living conidiospores of the naturally occuring B.bassiana strain ATCC 74040, isolated from Anthonomus grandis (Boheman), the cotton boll weevil, in the Lower Rio Grande valley, Texas, USA (BCPC, 2004). The formulated product  contains  at  least  2.3  x  107  viable  spores  /  ml.  Since  the  bioinsecticide  is  a dispersion of living conidiospores in vegetable oil, it can be used also in organic farming. In 2005,  Intrachem  Bio  International  S.A.  (Geneva,  Switzerland)  acquired  the  intellectual property  of  Naturalis  from  Troy  Biosciences,  and  the  commercial  product  is  now manufactured in Europe by Intrachem Production S.r.l (Grassobbio, Italy). B. bassiana strain ATCC 74040 acts primarily by contact: once attached to the insect’s cuticle,  the  conidiospores  germinate  producing  penetration  hyphae,  which  enter  and proliferate inside the insect’s body. The fungus feeds on its host, causing its death due to dehydration and/or depletion of nutrients.                                                 1 Edith Ladurner, Intrachem Production S.r.l., R & D Department, Italy – 47023 Cesena, edith.ladurner@intrachem.com 2 Massimo Benuzzi, Intrachem Bio Italia S.p.A., R & D Department, Italy – 47023 Cesena, massimo.benuzzi@intrachem.com 3 Fabio Fiorentini, Intrachem Production S.r.l., R & D Department, Italy – 47023 Cesena, fabio.fiorentini@intrachem.com 4 Sergio Franceschini, Intrachem Production S.r.l., Italy – 24050 Grassobbio, sergio.franceschini@intrachem.com Archived at http://orgprints.org/13651/94Infection can take between 24 and 48 hours, depending on the temperature (BCPC, 2004). The proliferation of the fungus inside the insect’s body leads to the insect’s death within 3-5 days.Several years of laboratory, semi-field and field studies showed that also Tephritid flies, Ceratitis capitata Wiedemann, Rhagoletis cerasi Loew, and Bactrocera oleae (Gmelin), are susceptible to infection by B. bassiana strain ATCC 74040 (Ladurner et al., 2007; Daniel etal., 2008). The European cherry fruit fly,  R. cerasi, is the major pest on sweet cherries throughout  Europe  and  in  parts  of  temperate  Asia  (Fischer-Colbrie  &  Busch-Petersen, 1989). If left uncontrolled, severe fruit damage may occurr, especially on medium to late ripening  cherry  varieties.  Furthermore,  R.  cerasi  control  is  becoming  increasingly challenging not only in organic and in integrated farming, but also in conventional production due to ecotoxicological and residue issues (Kovanci & Kovanci, 2006), and microbial control agents may thus represent valuable tools for the containment of this pest. The two trials conducted in 2004-05 aimed at verifying the efficacy of the B. bassiana-based bioinsecticide Naturalis, applied both alone and in an integrated pest management strategy, in controlling the cherry fruit fly on sweet cherry in the open field.Material and Methods The trials were conducted in 2004-2005 by a certified testing facility (G.Z. S.r.l., Italy) in Pavullo  (Modena,  Italy)  in  a  5-6-year  old  cherry  cv  Durone  Nero  orchard.  Distance between rows and plants was 4 m, and plant height was approximately 2.5 m. The efficacy in  reducing  the  percentage  of  fruits  damaged  by  R.  cerasi  of  the  B.  bassiana-based bioinsecticide  Naturalis,  applied  alone  at  two  different  rates  (130  and  250  ml/hl, respectively) and in an integrated pest management strategy, was compared to that of an untreated control and of a chemical reference insecticide. The different treatments, the number of applications per treatment, and the application dates are reported in Table 1.Table 1: Treatments, number of applications per treatment, and applications dates in the trials conducted in 2004-2005 (applied spray volume: 1500 l/ha).  Treatment no. Active substance  Formulated product (% a.i.) Appliedrate (ml/hl) No.applic.s Application dates (dd/mm) Trial no. 1-2004 1 B. bassiana strain ATCC 74040 Naturalis (7.16%)  130  5  28/05, 04/06, 11/06, 17/06, 23/062 B. bassiana strain ATCC 74040 Naturalis (7.16%)  250  3  28/05, 11/06,  23/063 Dimethoate + B. bassiana strain ATCC 74040Rogor L40 (38%) +  Naturalis (7.16%) 50 + 1301 + 228/0517/06, 23/06 4  Dimethoate  Rogor L40 (38%)  50  1  04/06 5  Untreated control  -  -  -  - Trial no. 2 - 2005 1 B. bassiana strain ATCC 74040 Naturalis (7.16%)  130  5  07/06, 13/06, 17/06, 22/06, 28/062 B. bassiana strain ATCC 74040 Naturalis (7.16%)  250  3  07/06, 17/06,  28/063 Dimethoate + B. bassiana strain ATCC 74040Rogor L40 (38%) +  Naturalis (7.16%) 50 + 1301 + 207/0622/06, 28/06 4  Dimethoate  Rogor L40 (38%)  50  1  13/06 5  Untreated control  -  -  -   Archived at http://orgprints.org/13651/95Treatments were applied from beginning of fruit colouring (BBCH 81; target stage: mostly eggs)  to  fruit  ripe  for  consumption  (BBCH  89;  target  stage:  mixed).  To  compare  the different  treatments,  a  fully  randomized  block  design  was  used  with  4  replicates  per treatment, and with 2 trees per plot. To assess percent fruit damage at harvest (28/06/04 and 30/06/05), in each plot, 5 kg of cherries were harvested, damaged and healthy fruits were counted, and the percentage of fruits damaged by R. cerasi was determined. Furthermore, the efficacy according to Abbott (1925) of the different treatments in reducing fruit damage at harvest was calculated.In each trial, the percentage of damaged fruits at harvest was compared across treatments using  one-way  ANOVA,  followed  by  the  Student-Newman-Keuls  test  for  posthoc comparison of means. Levene’s test was used to test for homogeneity of variances. ResultsIn both trials significant differences among treatments in the percentage of fruits damaged by the cherry fruit fly emerged (Table 2): fruit damage was always significantly higher in the  untreated  control  plots  than  in  the  treated  plots.  In  2004  no  significant  differences among treated plots were recorded, while in 2005 fruit damage was significantly lower in the plots treated with Naturalis at 130 ml/hl, with the IPM strategy, and in those treated with the reference insecticide dimethoate than in those treated with Naturalis at 250 ml/hl.  The mean efficacy of Naturalis applied  at 130 ml/hl ranged from 87 to 90%, and was always comparable to that of the IPM strategy (80-89%), and higher than or comparable to that of the chemical reference product applied alone (39-90%) (Table 2). Table 2: Percent fruit damage in the different treatments and trials and efficacy according to Abbott of the different treatments in reducing fruit damage at harvest. Different letters within the same column and trial indicate statistically significant differences (SNK-test: P<0.05). Treatment no. Active substance  Appliedrate (ml/hl) No.applic.s Fruit damage (%) (m±s.d.) Efficacy (%) (m±s.d.) Trial no. 1-2004 1 B. bassiana strain ATCC 74040 130  5  0.6 ± 0.6 b  88.6 ± 11.2 2 B. bassiana strain ATCC 74040 250  3  1.5 ± 0.8 b  72.9 ± 16.7 3 Dimethoate + B. bassiana strain ATCC 7404050 + 1301 + 2 1.2 ± 0.4 b  80.4 ± 5.4 4  Dimethoate  50  1  3.2 ± 1.6 b  39.0 ± 36.9 5  Untreated control  -  -  6.1 ± 2.0 a  - Trial no. 2 - 2005 1 B. bassiana strain ATCC 74040 130  5  1.3 ± 0.7 c  90.4 ± 5.1 2 B. bassiana strain ATCC 74040 250  3  4.4 ± 1.4 b  66.6 ± 9.9 3 Dimethoate + B. bassiana strain ATCC 7404050 + 1301 + 2 1.6 ± 1.2 c  88.5 ± 8.2 4  Dimethoate  50  1  1.2 ± 1.0 c  90.6 ± 8.4 5  Untreated control  -  -  13.2 ± 1.3 a   Archived at http://orgprints.org/13651/96Discussion In none of the trials a significant dose response effect was observed for the B. bassiana-based bioinsecticide. In both trials, instead, the efficacy of the product applied alone in reducing fruit damage was higher when it was used at a low rate (130 ml/hl) at 5-7-day intervals (5 applications in total) than when it was applied at a higher rate (250 ml/hl) at 10-14-day intervals (3 applications in total). Germination ability and spore viability of different B. bassiana strains are strongly linked to their tolerance to UV exposure (Bidochka et al.,2002; Fernandes et al., 2007), and conidial survival in epigeal habitats decreases over time due to exposure to UV-radiation (Inglis et al., 1993, 1995; Ulevicius et al., 2004). The results indicate that, for an effective control of this target pest, not exceeding the time interval of 7 days between successive applications is more important than increasing the field rate of the bioinsecticide. In order to efficiently control R. cerasi with Naturalis alone, the  product  should  thus  be  applied  from  the  beginning  of  fruit  colouring  up  to  harvest (period  of  highest  susceptibility  of  sweet  cherry  to  cherry  fruit  fly  attack),  at  a recommended  rate  of  approx.  130  ml/hl  and  at  a  5-7-day  time  interval  between applications.In 2004, the IPM strategy (one application of the standard reference product followed by two  applications  of  Naturalis  at  130  ml/hl  at  a  6-7-day  interval  just  prior  to  harvest) provided  good  and  consistent  control  of  R.  cerasi  (efficacy:  80.4±5.4%),  while  the dimethoate-based product showed high variability in suppressing cherry fruit fly damage (efficacy: 39±36.9%). In this trial, for the chemical reference treatment, the time interval between last application and harvest was 24 days. Pre-harvest interval instructions for the active ingredient dimethoate were followed (20 days on cherry in Italy in 2004-05), but cherry fruit fly control was highly variable among plots, and can thus not be considered satisfactory. This is probably due to the fact that close to harvest dimethoate levels on the vegetation  were  not  sufficient  any  more  to  provide  adequate  and  consistent  crop protection.In 2005, the efficacy of the chemical reference treatment was comparable to that of the IPM strategy, but pre-harvest interval requirements were not met (time interval between last application and harvest: 17 days), which translates to a high probability of undesired residues in fruit. Naturalis, instead, does not have any pre-harvest interval restrictions and minimum detectable residue level. The bioinsecticide can therefore be used up to harvest without any risk of undesired residues in the final production. In conclusion, the B. bassiana-based bioinsecticide, applied both alone and in an IPM strategy, showed high efficacy in reducing R. cerasi fruit damage on sweet cherry. The product can be considered an important and valuable tool for the control of this target pest not only in organic farming but also in any other pest management strategy, because it shows good efficacy in suppressing cherry fruit fly infestations and because it can help to avoid the presence of inadequate levels of residues in fruit.References Abbott, W.S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267.  BCPC (2004). Beauveria bassiana biological insecticide (fungus). In The Manual of Biocontrol Agents(ed. Copping, L.G.) pp. 43-46. Hampshire: BCPC.  Bidochka, M.J., Menzies, F.V. & Kamp, A.M. (2002). Genetic groups of the insect-pathogenic fungus Beauveria  bassiana  are  associated  with  habitat  and  thermal  growth  preferences.  Archives  of Microbiology 178: 531-537. Archived at http://orgprints.org/13651/97Daniel,  C.,  Keller,  S.  &  Wyss,  E.  (2008).  Field  applications  of  entomopathogenic  fungi  to  control Rhagoletis cerasi Loew (Diptera: Tephritidae). Insect Pathogens and Insect Parasitic Nematodes, COST 862, Bacterial Toxins for Insect Control, IOBC/wprs Bulletin, in press. Fernandes, E.K., Rangel, D.E., Moraes, A.M., Bittencourt, V.R. & Roberts, D.W. (2007). Variability in tolerance to UV-B radiation among Beauveria spp. isolates. Journal of Invertebrate Pathology 96:237-243. Fischer-Colbrie, P & Busch-Petersen, E. (1989). Pest status: temperate Europe and West Asia. In World Crop Pests: Fruit Flies, Biology, Natural Enemies and Control (ed. Robinson, A.S. & Hooper, G.H.S.) pp. 91-99. Amsterdam: Elsevier Science Publishers. Inglis,  G.D.,  Goettel,  M.S.  &  Johnson,  D.L.  (1993).  Persistance  of  the  entomopathogenic  fungus Beauveria bassiana on phylloplanes of crested wheatgrass and alfalfa. Biological Control 3: 258-270. Inglis,  G.D.,  Goettel,  M.S.  &  Johnson,  D.L.  (1995).  Influence  of  ultraviolet  light  protectants  on persistance of the entomopathogenic fungus Beauveria bassiana. Biological Control 5: 581-590. Knauf,  T.A.  (1992)  Naturalis-L:  a  biorational  insecticide  for  boll  weevil  and  whitefly  control.  Proc.Beltwide Cotton Conference 1: 21-32. Kovanci, O.B. & Kovanci, B. (2006). Reduced-risk management of Rhagoletis cerasi flies (host race Prunus) in combination with a preliminary phenological model. Journal of Insect Science 6: 34, available online: insectscience.org/6.34. Lacey, L.A., Horton, D.R., Chauvin, R.L. & Stocker, J.M. (1999). Comparative efficacy of Beauveria bassiana, Bacillus thuringiensis, and aldicarb for control of Colorado potato beetle in an irrigated desert agroecosystem and their effects on biodiversity. Entomologia Experimentalis et Applicata93: 189-200. Ladurner, E., Benuzzi, M. & Fiorentini, F. (2007). Un fungo per combattere i fitofagi che attaccano l’olivo. AZBIO 12: 52-56. Mayoral, F., Benuzzi, M. & Ladurner, E. (2006). Efficacy of the  Beauveria bassiana strain ATCC 74040 (Naturalis®) against whiteflies on protected crops. Integrated Control in Protected Crops, Mediterranean Climate, IOBC/wprs Bulletin 29: 83-88. Talaei-Hassanloui, R., Kharazi-Pakdel, A., Goettel, M.S., Little, S. & Mozaffari, J. (2007). Germination polarity  of  Beauveria  bassiana  conidia  and  its  possible  correlation  with  virulence.  Journal  of Invertebrate Pathology 94: 102-107. Ulevicius, V., Peciulyte, D., Lugauskas, A. & Andriejausiene, J. (2004). Field study on changes in viability of airborne fungal propagules exposed to UV radiation. Environmental Toxicology 19: 437-441. Discussion In none of the trials a significant dose response effect was observed for the B. bassiana-based bioinsecticide. In both trials, instead, the efficacy of the product applied alone in reducing fruit damage was higher when it was used at a low rate (130 ml/hl) at 5-7-day intervals (5 applications in total) than when it was applied at a higher rate (250 ml/hl) at 10-14-day intervals (3 applications in total). Germination ability and spore viability of different B. bassiana strains are strongly linked to their tolerance to UV exposure (Bidochka et al.,2002; Fernandes et al., 2007), and conidial survival in epigeal habitats decreases over time due to exposure to UV-radiation (Inglis et al., 1993, 1995; Ulevicius et al., 2004). The results indicate that, for an effective control of this target pest, not exceeding the time interval of 7 days between successive applications is more important than increasing the field rate of the bioinsecticide. In order to efficiently control R. cerasi with Naturalis alone, the  product  should  thus  be  applied  from  the  beginning  of  fruit  colouring  up  to  harvest (period  of  highest  susceptibility  of  sweet  cherry  to  cherry  fruit  fly  attack),  at  a recommended  rate  of  approx.  130  ml/hl  and  at  a  5-7-day  time  interval  between applications.In 2004, the IPM strategy (one application of the standard reference product followed by two  applications  of  Naturalis  at  130  ml/hl  at  a  6-7-day  interval  just  prior  to  harvest) provided  good  and  consistent  control  of  R.  cerasi  (efficacy:  80.4±5.4%),  while  the dimethoate-based product showed high variability in suppressing cherry fruit fly damage (efficacy: 39±36.9%). In this trial, for the chemical reference treatment, the time interval between last application and harvest was 24 days. Pre-harvest interval instructions for the active ingredient dimethoate were followed (20 days on cherry in Italy in 2004-05), but cherry fruit fly control was highly variable among plots, and can thus not be considered satisfactory. This is probably due to the fact that close to harvest dimethoate levels on the vegetation  were  not  sufficient  any  more  to  provide  adequate  and  consistent  crop protection.In 2005, the efficacy of the chemical reference treatment was comparable to that of the IPM strategy, but pre-harvest interval requirements were not met (time interval between last application and harvest: 17 days), which translates to a high probability of undesired residues in fruit. Naturalis, instead, does not have any pre-harvest interval restrictions and minimum detectable residue level. The bioinsecticide can therefore be used up to harvest without any risk of undesired residues in the final production. In conclusion, the B. bassiana-based bioinsecticide, applied both alone and in an IPM strategy, showed high efficacy in reducing R. cerasi fruit damage on sweet cherry. The product can be considered an important and valuable tool for the control of this target pest not only in organic farming but also in any other pest management strategy, because it shows good efficacy in suppressing cherry fruit fly infestations and because it can help to avoid the presence of inadequate levels of residues in fruit.References Abbott, W.S. (1925). A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18: 265-267.  BCPC (2004). Beauveria bassiana biological insecticide (fungus). In The Manual of Biocontrol Agents(ed. Copping, L.G.) pp. 43-46. Hampshire: BCPC.  Bidochka, M.J., Menzies, F.V. & Kamp, A.M. (2002). Genetic groups of the insect-pathogenic fungus Beauveria  bassiana  are  associated  with  habitat  and  thermal  growth  preferences.  Archives  of Microbiology 178: 531-537. Archived at http://orgprints.org/13651/

Beauveria bassiana strain ATCC 74040 (Naturalis®), a valuable tool for the control of the cherry fruit fly (Rhagoletis cerasi)

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