1. Introduction
The codling moth,
Cydia pomonella L. (Lepidoptera, Tortricidae), is a major and nearly worldwide occurring pest insect, mainly of apple trees, necessitating regular control measures. Generally, chemical insecticides are used, but also biological control methods, like pheromone mating disruption, as well as the specific
C. pomonella granulovirus (CpGV), are applied [
1,
2,
3,
4,
5,
6]. Concerns on safety and the environmental impact of insecticides, as well as the development of resistance have led to an increased use of CpGV. However, recently, some codling moth populations also showed a reduced susceptibility against CpGV products, stimulating the search for further biological control measures [
7,
8,
9,
10,
11,
12,
13,
14,
15,
16].
Several naturally occurring microbial antagonists have been reported from
C. pomonella, including viruses, bacteria, fungi, microsporidia and nematodes [
12,
17,
18,
19,
20]. So far, CpGV, the bacterium,
Bacillus thuringiensis, the fungus,
Beauveria bassiana, the microsporidium,
Nosema carpocapsae, and, recently, also, nematodes of the genus,
Steinernema, have been tested in the lab or in the field [
7,
8,
12,
15,
21,
22,
23,
24,
25,
26,
27,
28,
29].
Although the codling moth is a key pest in apple orchards, only a few ecological investigations on naturally occurring antagonists, especially insect pathogenic microorganisms, and on their importance as mortality factors in codling moth populations in Germany or Europe were conducted [
7,
18,
25,
30,
31,
32,
33,
34]. In this context, only a few studies on the causes of mortalities of diapausing larvae during hibernation are available, whereby pathogens could play an important role [
34,
35]. For example, Zelger
et al. [
36] found a winter mortality of 20–37%, mainly caused by weather conditions and general biotic factors, which are not clearly defined.
Recently, a review and a database on entomopathogens of insects and other arthropods found during long-term diagnostic studies at the Institute for Biological Control, Darmstadt, were published [
37,
38]. The data reported herein summarizes the type and frequency of pathogens found in 20,550 living, diseased or dead codling moth individuals that were diagnostically examined and the entomopathogens determined (E. Müller-Kögler, fungi: 1955–1974; A. Krieg, bacteria, rickettsiae, viruses: 1955–1989; A.M. Huger, viruses, bacteria, rickettsiae, microsporidia, protists: 1957–1991; G. Zimmermann, fungi: 1974–2005; R.G. Kleespies, viruses, bacteria, rickettsiae, microsporidia, protists: since 1991). As most of the results of these studies are not published and only few of them are briefly outlined in German annual reports of the former Federal Biological Research Centre for Agriculture and Forestry (BBA), a detailed presentation on
C. pomonella is given here. We believe that these results gained over a long period of time will stimulate further ecological and pathological studies on
C. pomonella, in order to enrich the understanding of the efficacy of natural microbial control factors within the population development. They also may initiate further approaches to preserve natural ecosystems and to develop new strategies within an integrated control system.
4. Discussion
The diagnostic studies represent an overview on entomopathogenic and other microorganisms, including nematodes, found in larvae, pupae and adults of the codling moth over a longer period of time. Except for N. carpocapsae, the investigations were not conducted systematically by routine examination of field populations, but irregularly whenever codling moth specimens were sent to the Institute for Biological Control for diagnosis of their diseases.
The most frequently diagnosed entomopathogens were the codling moth granulovirus (CpGV), various fungi, especially B. bassiana and I. farinosa, and N. carpocapsae. Long-term prognostic studies on the prevalence of N. carpocapsae in larvae and adults in various areas of Germany document the state and development of infestation in different field populations.
CpGV was only found in larvae and pupae from laboratory colonies and in field populations where the virus was applied for plant protection. The reason for the occurrence of CpGV in laboratory reared larvae is not always clear. Probably, they are contaminated accidentally by handling. Thus, a natural occurrence of CpGV, so far, is not proven in Germany, Austria or Switzerland. Certainly, it is difficult to find infected larvae in the field, as they rapidly decay. Whether a propagation of CpGV from virus-treated orchards to other untreated populations occurs, e.g., by contaminated flying insects, by birds or by atmospheric conditions, is scarcely investigated [
62,
63,
64]. The first detection of CpGV was in 1963 in an orchard in Mexico [
65].
The role of the observed unidentified bacteria, spore-formers or
Serratia spp. is unclear. They may be part of the normal gut flora, but some of them also may be pathogens. During epizootics in different laboratory colonies of
C. pomonella in Poland, several strains of the well-known pathogen,
B. thuringiensis, were isolated [
20]. In the present study, however,
B. thuringiensis was not found. In any case, further investigations on the effect of these microorganisms on individuals and on field populations of
C. pomonella are necessary.
Entomopathogenic fungi and, especially,
B. bassiana and
I. farinosa, were the major and most frequent group of pathogens diagnosed in last instars and diapaused larvae and pupae. The frequent occurrence and the high prevalence of
B. bassiana show that this species is an important naturally occurring mortality factor, mainly in hibernating populations, which obviously is permanently present in orchard ecosystems. This fungus has often been found in
C. pomonella and other tortricids [
7,
18,
30,
31,
32,
34,
66]. In an orchard in Austria (Steiermark), Russ [
31] observed an outbreak of
B. bassiana in diapausing larvae with prevalences of 77%, while in other more dry locations, only 1.3% and 2.5% of the larvae were infected. During investigations on diapausing larvae and in pupae of the codling moth in the south of Sweden, besides different parasitoids, mainly
B. bassiana (34.4%) and
I. farinosa (29.5%) were detected [
34]. In Nova Scotia, Canada, however, Jacques and MacLellan [
66] found that fungi only killed 1.7%, with a maximum of 10% of diapausing larvae of
C. pomonella populations. According to our knowledge, no detailed investigations on the role of entomopathogenic fungi as natural mortality factors and their impact on field populations in Germany and other European countries are available. Furthermore, it is unknown where and when larvae of the codling moth become infected by
B. bassiana or
I. farinosa in apple trees,
i.e., also studies on the ecology of these two fungi in the field are necessary to clarify their role as natural mortality factors. In addition, investigations on these fungi as pathogens of codling moth larvae from conventionally and ecologically treated apple orchards of different regions would be important to determine possible impacts of other plant protection treatments (e.g., fungicides or herbicides) on the prevalence of these antagonists.
The most frequently identified pathogen of codling moth larvae and adults was
N. carpocapsae, which causes a chronic disease. For the first time, data are presented on the occurrence and prevalence of
N. carpocapsae in
C. pomonella populations from various locations of Germany over a long period of time. It was demonstrated that this microsporidium causes prevalences of living specimens up to
ca. 70%. The average larval infestation levels in various codling moth populations from different areas in Germany (Hessen, Bayern, Baden-Württemberg) ranged from about 20% to 50% (see, also, [
25,
33]), which documents a relatively prevalent occurrence at that time. Similar infection levels were ascertained in adults caught in light traps in hessen over 18 years. This microsporidium was already observed in Germany in 1955 and, then, as documented here, in 1972 and in the following years in larvae, pupae and, particularly, also, in adults of
C. pomonella (see, also, [
33,
57]). This is remarkable, as
N. carpocapsae was reported for the first time in North America only in 2001 [
8]. Huger [
25,
33] demonstrated that the chronic infection is transmitted transovarially from infected females to the progeny. There was no significant difference in the infestation rate of female and male adults by
N. carpocapsae [
67].
Significant differences in the fecundity and fertility of healthy and infected adults of
C. pomonella were observed (
Figure 1; [
25]). Thus, in the infected batch, an average decrease of the number of hatched egg larvae by 56% was determined. Therefore, it can be concluded that, depending on the natural prevalence,
N. carpocapsae also may have an appreciable impact in reducing field populations. However, data on the actual infestation rates of codling moth populations by this microsporidium are not available. Similar observations have been published by Andreadis [
68] and Lewis
et al. [
69] for populations of the European corn borer,
Ostrinia nubilalis, infected by
Nosema pyrausta, resulting in a reduced fertility between 30% and 50%.
Reduced fertility of adults caused by
N. carpocapsae may also lead to some problems in laboratory reared colonies [
25,
70]. In contrast, Siegel
et al. [
8] did not find any effect of a North American isolate of
N. carpocapsae on the fecundity of females; however, a higher mortality and longer developmental period compared to healthy larvae and pupae were noticed. Furthermore, they showed that the first hatched larvae from one egg mass nearly were uninfected. Negative effects of microsporidia on the rate of progeny were also documented for the European corn borer,
O. nubilalis infected by
N. pyrausta [
71] and the gypsy moth,
Lymantria dispar, infected by
Vairimorpha disparis and
Nosema lymantriae [
72,
73,
74,
75,
76], where an increase in the mortality of larvae was noted. No effect of
N. carpocapsae on the mortality of diapausing larvae of
C. pomonella was observed [
33], while there was a clear correlation between the mortality of diapausing larvae of
O. nubilalis and their infestation by
N. pyrausta [
77].
In addition to these direct effects of
N. carpocapsae on its host,
C. pomonella, various interactions between this microsporidium and other antagonists are known. Experiments on the co-occurrence of
N. carpocapsae and CpGV in laboratory colonies revealed that microsporidia-infected individuals are six-times less susceptible to the CpGV compared to healthy ones [
78]. The effects of such double infections to codling moth populations under field conditions are unknown. Generally, microsporidia are rather selective entomopathogens with a small host range. However, in a laboratory colony of the codling moth parasitoid,
Ascogaster quadridentata (Hymenoptera, Braconidae), it was observed that
N. carpocapsae not only infects
C. pomonella, but also the egg-parasitoid itself, thus resulting in a collapse of the rearing [
79,
80]. Furthermore, an infection of the egg-parasitoid,
Trichogramma evanescens, by
N. carpocapsae was proven, causing a reduction of the parasitic capacity of the microsporidia-infected
Trichogramma females [
81]. Possible interactions of
N. carpocapsae with these parasitoids and their effect on codling moth populations in the field and their ecological relevance are not yet investigated.
Diagnostic studies on the occurrence and prevalence of naturally occurring insect pathogens of
C. pomonella are important, also with regard to their possible use as biocontrol agents. For example, a strain of
M. anisopliae (M.a. 43, JKI fungus collection) isolated from a codling moth larva in 1971 originating from Austria was successfully used under the strain names, F52 or BIPESCO 5, against other pest insects, e.g., the black vine weevil,
Otiorhynchus sulcatus (Coleoptera: Curculionidae), and was the active ingredient of the former mycoinsecticide, “BIO 1020”, of the company, BAYER [
82] or against white grubs of the garden chafer [
83].
So far, only the CpGV is commercialized for codling moth control (e.g., [
3]), but recently, also, entomoparasitic nematodes (
Steinernema carpocapsae and
S. feltiae) were tested successfully [
7,
15,
27,
29,
84,
85]. Formerly, also, the fungus,
B. bassiana, was tested in laboratory and field experiments [
7,
21,
23,
24,
26]. As already suggested [
21] and according to our findings of the fungus in the last larval or diapausing instars, an application on branches and stems in late summer or autumn seems to be most promising, as well as a soil application under trees to infect overwintering larvae and to increase the antagonistic potential. In contrast to these insect pathogens,
N. carpocapsae was never used as a biocontrol agent against
C. pomonella. However, other microsporidia, such as
Nosema pyrausta, were successfully introduced in field populations of the European corn borer,
Ostrinia nubilalis [
68,
86,
87,
88]. Furthermore, the microsporidia, Nosema sp. and Vavraia sp., could be established in populations of the gypsy moth,
Lymantria dispar [
89].