In a pilot field study conducted in an apple orchard in Hardanger (Western Norway) in two

succesive years both Beauveria bassiana and Metarhizium anisopliae were applied to

apple trees just after hatching of mirid nymphs in spring. Both predatory, omnivorous and

principally plant-pathogenic species of mirids were collected, and all groups of species

were infected by B. bassiana or M. ansiopliae in treated plots. B. bassiana and M.

anisopliae were also found on some individuals in non treated control plots the second

year. M. anisopliae was more predominant in 2006, B. bassiana in 2007. Mirids were also

observed for natural occurrence of parasitoids, and parasitoids were found both years

Hovland, B.

Jaastad, G.

Klingen, I.

Westrum, K.

English

Organic Eprints

186Pilot field studies on insect pathogenic fungi to control mirid pests of apples in Norway G. Jaastad1, I. Klingen2, K. Westrum2 and B. Hovland1AbstractIn a pilot field study conducted in an apple orchard in Hardanger (Western Norway) in two succesive years both Beauveria bassiana and Metarhizium anisopliae were applied to apple trees just after hatching of mirid nymphs in spring. Both predatory, omnivorous and principally plant-pathogenic species of mirids were collected, and all groups of species were  infected  by  B.  bassiana  or  M.  ansiopliae  in  treated  plots.  B.  bassiana  and  M.anisopliae were also found on some individuals in non treated control plots the second year. M. anisopliae was more predominant in 2006, B. bassiana in 2007.  Mirids were also observed for natural occurrence of parasitoids, and parasitoids were  found both years.Keywords:  Beauveria  bassiana, Metarhizium  anisopliae,  biological  control,  insect pathogenic fungi, Miridae, Norway, parasitoids IntroductionInsect pathogenic fungi have shown to be effective in controlling important insect pests both in conservation biological control and as bio-based insecticides (Copping, 2004; de Faria & Wright, 2007). Earlier work has shown that several species of insect pathogenic fungi can cause death in mirids (Hemiptera: Miridae) (Liu  et al., 2003; McGuire  et al.,2006; de Faria & Wright, 2007).Mirids are important pests in apple and pear production in both Europe and the United States (Wheeler, 2001; Alford, 2007). In organic production the degree of fruit damage can exceed 40 % (Røen et al., 2003). Mirid bugs feed on fruit sap in shoot tips, flower buds and fruitlets, resulting in deformation and stony pits in the fruit (Wheeler, 2001). Mirids damaging fruit are usually both polyphagous and omnivorous, and it can be difficult to assess whether a species should be considered as a pest or as a beneficial insect. To be able to use insect pathogenic fungi for the biocontrol of mirids it is important to evaluate their effects on fruit damage as well as against various species of mirids.The  objective  of  this  study  was  to  conduct  a pilot  study  to  evaluate  the  effect  of  field application of Norwegian isolates of the two insect pathogenic fungi Beauveria bassiana(NCRI  12/96)  and  Metarhizium  anisopliae  (NCRI  250/02)  on  (1)  mortality  and  fungal growth in the most abundant mirid species found in the field, and (2) mirid damage on fruit. Material and Methods Trials  were  conducted  over  two  successive  years  in  an  apple  orchard  in  Hardanger, (Western Norway). Two different fields in the same orchard were used in 2006 and 2007. In both years experiments were set up as a randomized block design with five replications, and in each plot there were 3 trees cv ‘Aroma’, and a minimum of 2 border trees between each plot.                                                1 Norwegian Institute for Agricultural and Environmental Research - Bioforsk Horticulture and Greening Division, Bioforsk Ullensvang, N-5781 Lofthus, Norway, e-mail: gunnhild.jaastad@bioforsk.no 2 Norwegian Institute for Agricultural and Environmental Research – Bioforsk Plant Health and Plant Protection, Høgskoleveien 7, N-1432 Ås, Norway Archived at http://orgprints.org/13673/187Pilot field studies on insect pathogenic fungi to control mirid pests of apples in Norway G. Jaastad1, I. Klingen2, K. Westrum2 and B. Hovland1AbstractIn a pilot field study conducted in an apple orchard in Hardanger (Western Norway) in two succesive years both Beauveria bassiana and Metarhizium anisopliae were applied to apple trees just after hatching of mirid nymphs in spring. Both predatory, omnivorous and principally plant-pathogenic species of mirids were collected, and all groups of species were  infected  by  B.  bassiana  or  M.  ansiopliae  in  treated  plots.  B.  bassiana  and  M.anisopliae were also found on some individuals in non treated control plots the second year. M. anisopliae was more predominant in 2006, B. bassiana in 2007.  Mirids were also observed for natural occurrence of parasitoids, and parasitoids were  found both years.Keywords:  Beauveria  bassiana, Metarhizium  anisopliae,  biological  control,  insect pathogenic fungi, Miridae, Norway, parasitoids IntroductionInsect pathogenic fungi have shown to be effective in controlling important insect pests both in conservation biological control and as bio-based insecticides (Copping, 2004; de Faria & Wright, 2007). Earlier work has shown that several species of insect pathogenic fungi can cause death in mirids (Hemiptera: Miridae) (Liu  et al., 2003; McGuire  et al.,2006; de Faria & Wright, 2007).Mirids are important pests in apple and pear production in both Europe and the United States (Wheeler, 2001; Alford, 2007). In organic production the degree of fruit damage can exceed 40 % (Røen et al., 2003). Mirid bugs feed on fruit sap in shoot tips, flower buds and fruitlets, resulting in deformation and stony pits in the fruit (Wheeler, 2001). Mirids damaging fruit are usually both polyphagous and omnivorous, and it can be difficult to assess whether a species should be considered as a pest or as a beneficial insect. To be able to use insect pathogenic fungi for the biocontrol of mirids it is important to evaluate their effects on fruit damage as well as against various species of mirids.The  objective  of  this  study  was  to  conduct  a pilot  study  to  evaluate  the  effect  of  field application of Norwegian isolates of the two insect pathogenic fungi Beauveria bassiana(NCRI  12/96)  and  Metarhizium  anisopliae  (NCRI  250/02)  on  (1)  mortality  and  fungal growth in the most abundant mirid species found in the field, and (2) mirid damage on fruit. Material and Methods Trials  were  conducted  over  two  successive  years  in  an  apple  orchard  in  Hardanger, (Western Norway). Two different fields in the same orchard were used in 2006 and 2007. In both years experiments were set up as a randomized block design with five replications, and in each plot there were 3 trees cv ‘Aroma’, and a minimum of 2 border trees between each plot.                                                1 Norwegian Institute for Agricultural and Environmental Research - Bioforsk Horticulture and Greening Division, Bioforsk Ullensvang, N-5781 Lofthus, Norway, e-mail: gunnhild.jaastad@bioforsk.no 2 Norwegian Institute for Agricultural and Environmental Research – Bioforsk Plant Health and Plant Protection, Høgskoleveien 7, N-1432 Ås, Norway The field used in 2006 was planted in 1994 and had a tree height of 2-2.5 m, whereas in 2007 a field planted in 1993 with a tree height 2.5-3 m was used. Both fields had a tree planting  distance  of  4.5  x  1.5  m.  In  both  years  no  pesticides  were  used  from  the application of pathogens onwards until 2 weeks after the last sampling of insects. The two isolates were grown in Petri dishes on potato dextrose agar (B. bassiana) and sabouraud dextrose agar (M. anisopliae) for 25 days at room temperature in the dark until good sporulation was obtained. About 15 ml autoclaved aqueous Tween solution (0.05%) was added to each Petri dish, followed by separation of spores from the mycelium with a sterile glass spatula. 25 ml of the spore suspension were then injected by a sterile syringe into a polypropylene bag (20 x 30 cm) with 200 g of Jasmine rice and 85 ml of water previously autoclaved for 5 minutes in 121 oC and cooled. Inoculated bags were stored at room temperature in the dark for at least 2 weeks and shaken gently every 2-3 days. B. bassiana and M. anisopliae growing on rize were diluted in water and 0.05% Tween, poured through a sieve (mesh size 1 mm) to  obtain a spore suspension  whithout  rize debris, and applied with a hand-held Hardy wheelbarrow sprayer until run-off. Samples from each suspension were collected to determine the applied spore concentration. Two applications were performed in both 2006 and 2007, the first just before flowering (first flower  open)  and  the  second  at  petal  fall.  Application  time,  application  volume  and concentration of spores (spores/ml and spores/ha) for both years are given in Table 1.Table 1. Application time, volume (l/100 m row) and spore concentration (spores per ml and per ha) for first and second applications of B. bassiana and M. anisopliae in 2006 and 2007. Year  Application time  Species  Spores/ml  l/ha  Spores/ha 2006  30 May  B. bassiana  6 x 106  1362  8 x 1012M. anisopliae  6 x 106  1778  1 x 1013  12 June  B. bassiana  5 x 106  1680  8 x 1012M. anisopliae  5 x 106  1778  1 x 1013     2007  21 May  B. bassiana  7 x 107  2549  2 x 1014M. ansiopliae  5 x 107  2351  1 x 1014  29 May  B. bassiana  2 x 107  2589  5 x 1013     M. anisopliae  3 x 107  2687  7 x 1013To investigate the incidence of fungal infection, insects were collected by beating three random branches on every tree in each plot with a stiff rubber hose both one and two weeks  after  the  last  spray  application.  Each  branch  received  three  quick  beats,  and dislodged insects were collected by a beating tray. All insects were identified and counted, but only mirids and Anthocoris nemorum (L.) were incubated. All mirids collected were nymphs, most of them in the third or fourth instar. Both adults and nymphs of A. nemorumwere collected. Individual mirids were placed on an apple shoot embedded in 1.5 % water agar in a 25 ml vial. Mirids were incubated at 20°C, 70% relative humidity and 16 hours light  in  both  years.  Mortality  was  recorded  daily?  for  seven  days.  Dead  mirids  were transferred to a new 25 ml vial with 1.5 % water agar for observation of fungal growth. Fungi were identified by observing characteristic fungal growth patterns on the mirids and by  microscopic  examination  (x600).  Mirids  were  also  examined  for  parasitoids  by observing for emerging larvae and by dissection of dead nymphs. Parasitoid species were not identified. Archived at http://orgprints.org/13673/188Data presented are numbers of infected mirids and A. nemorum in the different treatments. An analysis of variance for mirid damage on fruits was done with treatment and block as explanatory variables (SAS Institute, 2003). ResultsIn treated plots mirids were found infected with B. bassiana and M. anisopliae in both years. In addition, some individuals in the control plots were infected in 2007 (Table 2 and 3). In 2006 only Lygocoris pabulinus (L.) and Psallus ambiguus (Fallén) were infected with B. bassiana, but only P. ambiguus also with M. anisopliae (Table 2). These two species were  also  the  most  abundant  mirids  in  2006.  A  higher  number  of  mirids  were  found infected in the first sampling compared to the second sampling (data not shown). In 2006, the number of mirids with naturally occurring parasitoids was higher than mirids infected with fungi, and both Orthotylus marginalis (Reuter), L. pabulinus and P. ambiguus were found with parasitoids (Table 2). Table 2: Total number of mirids and Anthocoris nemorum (N), and number of individuals infected by Beauveria bassiana, Metarhizium anisopliae or found with parasitoids in different treatments in 2006. Data from first and second samplings are combined. Treatment Species NNumber with B. bassiana Number with M. anisopliae Number with parasitoids Control Lygocoris pabulinus  26  0  0  2 Control Orthotylus marginalis  2  0  0  1 Control Psallus ambiguus  17  0  0  6 Control Phytocoris sp. 9  0  0  0 Control Anthocoris nemorum  30  0  0  0 Beauveria  Lygocoris pabulinus  14  0  0  0 Beauveria  Orthotylus marginalis  0  -  -  - Beauveria  Psallus ambiguus  16  4  0  1 Beauveria Phytocoris sp. 18  0  0  0 Beauveria  Anthocoris nemorum  30  0  0  0 Metarhizium  Lygocoris pabulinus  21  0  1  2 Metarhizium  Orthotylus marginalis  4  0  0  1 Metarhizium  Psallus ambiguus  25  0  7  4 Metarhizium Phytocoris sp. 18  0  0  0 Metarhizium  Anthocoris nemorum  26  0  0  0 The degree of infection by B. bassiana and M. anisopliae was generally higher in 2007 than in 2006 (Table 3). All of the presented insect species were found to be infected with both fungi in 2007. A B. bassiana infection was found in plots treated with M. anisopliae,and in the second sampling infection by B. bassiana was higher than M. anisopliae in plots treated with M. anisopliae (data not shown). One species, P. ambiguus, was found to be infected with M. anisopliae in plots treated with B. bassiana. Further, infections with both pathogens were found in the control. The number of insects carrying parasitoids was low in 2007, only P. ambiguus parasitism. Treatment with the selected entomopathogens did not reduce crop damage by mirids in this study. Neither treatment nor block had a significant effect on damage in either year (Table 4) (2006: df = 6, 8, F = 1.14, p = 0.42; 2007: df = 6,8, p = 1.47, p = 0.30). Archived at http://orgprints.org/13673/189Table  3:  Total  number  of  mirids  and  Anthocoris  nemorum,  and  number  infected  by  Beauveriabassiana, Metarhizium anisopliae or found with parasitoids in different treatments in 2007. Data from first and second samplings are combined. Treatment Species NNumber with B. bassiana Number with M. anisopliae Number with parasitoids Control Lygocoris pabulinus  5  1  0  0 Control Orthotylus marginalis  4  0  0  0 Control Psallus ambiguus  35  3  0  2 Control Phytocoris sp. 11  0  1  0 Control Anthocoris nemorum  9  0  0  0 Beauveria  Lygocoris pabulinus  7  2  0  0 Beauveria  Orthotylus marginalis  6  3  0  0 Beauveria  Psallus ambiguus  20  11  1  0 Beauveria Phytocoris sp. 12  9  0  0 Beauveria  Anthocoris nemorum  12  3  0  0 Metarhizium  Lygocoris pabulinus  0  -  -  - Metarhizium  Orthotylus marginalis  8  4  1  0 Metarhizium  Psallus ambiguus  36  3  11  5 Metarhizium Phytocoris sp. 9  0  1  0 Metarhizium  Anthocoris nemorum  15  3  4  0 Table 4: Percent capsid damage on apples at harvest in 2006 and 2007. 100 apples per plot were investigated for damage (n = 5). Year  Treatment  Total number of apples  Average % damage 2006  Control  500  1.4   B. bassiana  500  2.0   M. anisopliae  500  1.0        2007  Control  500  19.4   B. bassiana  500  15.8   M. anisopliae  500  13.0 Discussion Both M. anisopliae and B. bassiana were found to infect the species of mirids collected in this trial. M. anisopliae was predominant in 2006, whereas in 2007 B. bassiana was more common. These differences might be caused by a higher concentration of M. anisopliaespores at both applications in 2006 and a higher concentration of B. bassiana spores in the first application in 2007. Generally, a higher degree of  infection and more species being infected in 2007 might be caused by a higher concentration of spores and a higher spray volume used in that year. Fungal infections were also found in control plots in 2007. This  might  indicate  a  natural  infection  of  B.  bassiana  and  M.  anisopliae  in  the  field, although a more likely explanation is that spores were transported by wind or water during spraying or by flying insects shortly after application. In future work, distance between plots should be increased to rule out these factors. More work is needed to investigate the natural fungal infection on mirids in Norway. Predatory species (A. nemorum and Phytocoris sp.), omnivorous species (O. marginalisand P. ambiguus) and clearly pest species (L. pabulinus) were infected in this study. As far as we know, previous work on the effect of B. bassiana and M. anisopliae on mirids has focused on pest species (Liu et al., 2003; McGuire et al, 2006).Archived at http://orgprints.org/13673/190However, to evaluate the net benefit of a pathogen as a control method against insects, the effect on both pests and beneficials are needed. The mirid P. ambiguus seem to be susceptible to both fungal isolates tested. Based on our data  it  also  seemed  to  be  more  attractive  to  naturally  occurring  parasitoids.  Both  P. ambiguus  and  O.  marginalis  are  mainly  regarded  as  predators,  but  might  occasionally cause damage because they are able to feed on plants (Taksdal, 1983; Wheeler, 2001; Edland, 2004). However, it is likely that P. ambiguus is causing fruit damage more often than assumed earlier. Unpublished data show that number of P. ambiguus is positively correlated  to  damage  in  apples  (G.  Jaastad,  N.  Trandem,  S.  Mogan  &  B.  Hovland, unpublished). Further, Taksdal (1983), showed that P. ambiguus can cause stony pits in pears in an isolation bag trial. Lastly, in this study more individuals of this species were found in 2007 (total of 93) compared to 2006 (total of 52), and more damage was recorded in 2007 compared to 2006.Treatment with B. bassiana and M. anisopliae did not affect the degree of damage caused by mirids in this study. Visual damage by mirids is dependent on the time-point of feeding during the phenological development of fruits. According to a review by Wheeler (2001) mirid feeding on flowers and fruitlets between the bloom stage and two weeks after petal fall is most likely to cause visible damage on apples. Thus, if mirids do not die shortly after they have become infected by fungi, damage might occur. In summary, this study shows that  both  beneficial,  omnivorous  and  pest  species  of  mirids  can  be  infected  by  M. anisopliae  and  B.  bassiana  after  field  application.  However,  a  clear  effect  of  pathogen infection on mirid damage to apples was not found. More work on insect pathogenic fungi and mirids in fruit orchards is needed to evaluate their potential effect as control agents against this group of pests. Acknowledgements We thank Sjur K. Jaastad for permission to conduct these experiments in his orchard, and Jostein Ulgenes and Rolf Tore Djønne for valuable assistance during field work. This work was financially supported by the Norwegian Research Institute. References Alford, D.V. (2007). Pests of Fruit Crops. Academic Press, Burlington, MA, USA. Copping, L.G. (2004). The Manual of Biocontrol Agents (third edition). BCPC, Hampshire, England. De Faria, M.R. & Wright, S.P. (2007). Mycoinsecticides and Mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types. Biological Control43: 237-256. Edland, T. (2004). Sugande skade- og nyttedyr i frukthagar [in Norwegian]. Grønn Kunnskap 8 (4).175 pp. Liu, H., Skinner, M., Brownbridge, M., Parker & Parker, B.L. (2003). Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug, Lyguslineolaris (Hemiptera: Miridae). Journal of Invertebrate Pathology 82: 139-147. McGuire, M.R., Leland, J.E., Dara, S., Park, Y-H. & Ulloa, M. (2006). Effect of different isolates of Beauveria bassiana on field populations of Lygus hesperus. Biological Control 38: 390-396. Røen,  D.,  Nornes,  L.  &  Jaastad,  G.  (2003).  Årsaker  til  fråsortering  i  økologisk  dyrka  frukt  [in Norwegian]. Norsk Frukt og Bær 6: 28-30 SAS Institute (2003). Version 9.1. SAS Institute Inc., Cary, NC, USA. Taksdal,  G.  (1983).  Orthotylus  marginalis  Reuter  and  Psallus  ambiguus  (Fallén)  (Heteroptera, Miridae) causing stony pits in pears. Acta Agriculturae Scandinavica 33: 205-208. Wheeler, A.G. (2001). Biology of the Plant Bugs. Cornell University Press, New York, USA. Archived at http://orgprints.org/13673/

Pilot field studies on insect pathogenic fungi to control mirid pests of apples in Norway

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