The bulb mite, Rhizoglyphus robini, is a serious pest of garlic, onion and other crops. The mite is usually found in association with dangerous fungal pathogens such as Fusarium spp. Control of this pest has relied upon the use of synthetic acaricides but chemical control of the bulb mite is difficult because it is able to develop resistance quickly. Thus, alternative control methods, e.g. biological control, need to be developed and implemented. The aim of this study was to assess efficacy of selected strains of entomopathogenic fungi (EPF) against adult females of R. robini under laboratory conditions. New EPF strains were isolated from soil samples collected in onion and garlic fields in the Czech Republic and Israel using soil elution and cultivation on selective media. Fungal species were determined using macroscopic, microscopic and molecular markers. The efficacy against R. robini females was tested in 17 isolated and 3 reference strains of EPF. Results revealed high variability among species and strains. The highest efficacy against R. robini mites was found in strains of Metarhizium anisopliae isolated from soil samples collected in the Czech Republic which caused mortality up to 99.3%, and a Metarhizium indigoticum strain from Israel causing 98.3% mortality after four days of bioassay. Isaria fumosorosea strains did not caused mortality higher than 40%. The lowest virulence was found in Beauveria spp. strains causing mortality of mites between 5 and 25%. Median lethal time (LT50) and median lethal concentration (LC50) in the three most virulent strains ranged between 2 and 4 days and between 1.01×104 and 2.36×105 spores/ml, respectively. The concentration-response models indicated that the M. indigoticum strain is more lethal than M. anisopliae strains. The present study showed that some strains of entomopathogenic fungi, especially from the genus Metarhizium, could be perspective biocontrol agents against R. robini.
Introduction
Bulb mites of the genus Rhizoglyphus are economically important pests of plants with bulbs, corns, and tubers. Their main hosts are species in the family Liliaceae but they often attack other important crops such as potatoes (Solanum sp.) and carrots (Daucus carota) (Díaz et al. 2000). Fan and Zhang (2004) published a revision of the Australasia and Oceana species of Rhizoglyphus and more recently Barbosa and Moraes (2020) reported on the species in Brazil.
Rhizoglyphus robini (Claparède) (Acari: Acaridae) is considered one of the most serious pests of onion, garlic and ornamentals such as lily, tulips and hyacinths in storage, greenhouse and in the field around the world (Díaz et al. 2000; Fan & Zhang 2004). This species has a very high reproductive rate. When offered peanuts as a sole food source, the mean total fecundity was 690 eggs and the intrinsic rate of population increase (rm) was estimated to be 0.285 (Gerson et al. 1983). Besides causing direct feeding damage, this pest also disseminates phytopathogenic bacteria and fungi, e.g. Fusarium oxysporum, which infect bulbs, facilitating the pathogens entry into host plants (Poe et al. 1979; Okabe & Amano 1991; Díaz et al. 2000; Hanuny et al. 2008; Zindel et al. 2013; Ofek et al. 2014).
With the exceptions of solarization of soil (Gerson et al. 1981) or hot-water treatment of bulbs (Conijn 1992), the control of this pest is still based almost entirely on broad-spectrum pesticides, even though it has been known for many years that bulb mites quickly develop resistance to pesticides (Poe et al. 1979; Kuwahara 1988; Díaz et al. 2000). Furthermore the application of these chemicals to soil negatively impacts non-target organisms, e.g. earthworms and soil microorganisms, leaves residues in food crops and contaminates groundwater. For these reasons broad-spectrum pesticides have been targeted by the European community for deregistration (EC 2009). Thus, alternative, environmentally safe control strategies, e.g. biological control, need to be developed and implemented. Efforts to develop biocontrol techniques for bulb mites have been undertaken in many countries and mostly involved the use of soil-dwelling predatory mites, e.g. Gaeolaelaps aculeifer (Canestrini) (Acari: Laelapidae) (Lesna et al. 1995, 1996). A recent study by Nermuť et al. (2019) showed that some, especially small, entomopathogenic nematodes (EPN) are able to invade and kill adult females of R. robini. The most promising species were Steinernema huense (Nematoda: Steinernematidae), Heterorhabditis bacteriophora and H. amazonensis (Nematoda: Heterorhabditidae) causing mortality in R. robini up to 30%. Mortality of mites treated by culture supernatants of the nematode symbiotic bacteria of the genus Xenorhabdus was generally lower but some bacterial strains showed repellent effect to mites. Due to their relatively low efficacy, EPNs and the metabolites of their symbiotic bacteria do not seem to represent a viable option for bulb mite biocontrol as a standalone approach (Nermuť et al. 2019).
Entomopathogenic fungi (EPFs) represent another promising group of biocontrol agents. Their advantages are that they do not need to be ingested as they are able to penetrate the host cuticle and can be relatively easily produced (Shahid et al. 2012). Many EPFs species attack Acari and can be used for biological control of mite pests. Besides Acari-specific pathogens such as Hirsutella thompsonii (Fisher) and Neozygites spp. (Entomophthorales), ‘nonspecialist’ mitosporic fungi (Hyphomycetes) like Beauveria bassiana (Bals.-Criv.) Vuill., Metarhizium anisopliae (Metsch.) Sorokin, Isaria fumosorosea (Wize), I. farinosa (Holmsk.), and Lecanicillium lecanii (Zimm.) Zare & W. Gams have potential to control some mite species (Chandler et al. 2000). Most studies on efficacy of EPFs against mites have targeted ticks (Kaaya et al. 1996; Kaaya & Hassan 2000; Fernandes & Bittencourt 2008), spider mites (Chandler et al. 2005; Wekesa et al. 2005; Shi & Feng 2009; Ullah & Lim 2017; Shang et al. 2018; Khoury et al. 2020) and eriophyoid mites (Latge et al. 1988; McCoy 1996; Van der Geest et al. 2000). To our knowledge, only three EPFs species have been tested against R. robini under laboratory or greenhouse conditions: Hirsutella kirchneri (Rostrup) Minter, Brady and Hall (Sztejnberg et al. 1997), I. fumosorosea (Zemek et al. 2018) and Metarhizium brunneum Petch (Ment et al. 2020). While the first two species were not able to control R. robini, M. brunneum was found to be a promising biocontrol agent against this pest.
The aim of our study was to assess the possibility of fungal biocontrol of R. robini by new EPF strains isolated from soil samples collected in onion and garlic fields in the Czech Republic and Israel. The efficacy of these strains was compared with I. fumosorosea strain CCM 8367 and two commercially used strains, B. bassiana strain GHA and M. brunneum Petch strain F52.
Material and methods
Rhizoglyphus robini
The laboratory culture of R. robini was established from mites originating from rotting onion plants collected in Israel. The mites were maintained in large Petri dishes lined with wet filter paper using crushed raw peanuts as a food source. The dishes with mites were kept in darkness at 20 °C.
Entomopathogenic fungi
In total, 17 EPF strains were isolated from soil of several, mostly pesticide free onion and garlic fields in Pilsen and South Bohemian regions in the Czech Republic and in the Beit She'an Valley, Jezreel Valley and Lower Galilee in Israel. Soil sampling was performed during vegetation season in 2017. EPF isolates were obtained by water elution of soil samples and cultivation using selective medium containing dodine (Chase et al. 1986). Strains of EPF were identified on the basis of macroscopic, microscopic and genetic characteristics.
DNA for genetic analysis was extracted from fresh mycelium grown at 25±1 °C for 7 days on Petri dishes with PDA (Sigma-Aldrich, Darmstadt, Germany) medium. Each mycelium was collected to a sterile 1.5 mL microtube. The extraction method used was based on CTAB-PVP (Doyle 1991) with modification for fungi. Genomic DNA was amplified by PCR with universal primers NL1 5′-GCATATCAATAAGCGGAGGAAAAG-3′ (forward) and NL4 5′-GGTCCGTGTTTCAAGACGG-3′ (reverse) (O'Donnell 1992; 1993). PCR reactions were carried out in a volume 25 µL containing in 1X reaction buffer (75 mM Tris–HCl, pH=8.8, 20 mM (NH4)2SO4, 0.01% Tween® 20 (Sigma-Aldrich, Darmstadt, Germany), 2.5 mM MgCl2, 200 µM dNTPs), 1.25 U Taq Purple DNA polymerase (PPP Master Mix, Top-Bio, CZ), 10 pmol of both forward and reverse primer and 50 ng template DNA. Microtubes were placed in a thermal cycler (TProfessional Basic Gradient, Biometra) with the following program: 1 cycle of 94 °C for 5 min, 25 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min and 15 s, and final elongation at 72 °C for 5 min. The part of amplified PCR products was visualized on 2% agarose gel. The PCR products were sequenced by SEQme (Czech Republic). The sequences obtained were edited, compiled and aligned using Geneious (New Zealand) software. Sequence similarity searches were performed using NCBI GenBank BLASTn.
Cultures have been deposited at the Biology Centre CAS, České Budějovice. GenBank accession numbers for all 17 strains are listed in Table 1. In addition, three reference strains were used in efficacy bioassays. Two reference strains were re-isolated from commercial mycopesticides: B. bassiana strain GHA (BotaniGard® WP, Certis USA, Llc., Butte, MT, USA) and M. brunneum strain F52 (Met52® EC, Novozymes Biological, Franklinton, NC, USA). The species M. brunneum was previously classified as M. anisopliae (Bischoff et al. 2009). The third strain was CCM 8367 strain of I. fumosorosea, which originates from the horse chestnut leaf miner, Cameraria ohridella, Deschka & Dimić (Lepidoptera: Gracillariidae) collected in the Czech Republic (Zemek et al. 2007). The strain is patented (Prenerová et al. 2013, 2015) and deposited in the Czech Collection of Microorganisms in Brno.
All strains were cultivated on PDA medium at 25±1 °C and 16L:8D photoperiod. After 10 days of incubation, the spore suspensions were prepared from each strain by scraping off conidiospores into a sterile solution of 0.05% (v/v) Tween® 80 (Sigma-Aldrich, Darmstadt, Germany). Suspensions were filtered through sterile gauze to separate the mycelium and clusters of spores. In uniform suspension, the number of spores was counted with a Neubauer improved counting chamber (Sigma-Aldrich, Darmstadt, Germany) and subsequently the suspension was adjusted to the required concentration. The suspension was left for approximately 12 hours at temperature 23±1 °C to accelerate and synchronize germination of conidia (Dillon & Charnley 1985, 1990) before its application. Viability of spores was verified using a standard germination test (Skalický et al. 2014). Ten drops from suspension were applied using a 1 µl inoculation loop on the surface of 2% water agar, which was poured in a thin layer onto the surface of a sterile slide. After the drops had dried, the slides were moved into a wet chamber and incubated at 25±1 °C for 24 h. Percentage of germinating spores was determined using an Olympus CH20 light microscope (Olympus Optical Co., Ltd., Tokyo, Japan); bright field, 400× magnification. The spore germination of all strains was >95%.
Bioassays
Efficacy of EPF strains was assessed in single-dose bioassays using 24-well polystyrene tissue culture plates (Orange Scientific, Braine-L'Alleud, Belgium). Plate dimensions were 128×86 mm and bottom area of single well was 193 mm2. Filter paper discs of 14 mm in diameter were placed into each well and moistened with 100 µL of sterile distilled water. Water did not only provided moisture, but also created a surface tension that prevented the mites from escaping from the experimental arena (Chen 1990). Four R. robini females were placed into each well and the plate was sprayed with 2 mL of fungus suspension with concentration 1×107 spores per 1 mL using a Potter spray tower (inner diameter of cylinder 29 cm, spray pressure 50 kPa). Density of conidia was thus approx. 3×104 spores per cm2, i.e. 5.8×104 spores per a single plate well. Control variant was treated with a sterile solution of 0.05% Tween 80®. After treatment, plates were covered with lids and incubated at 25±1°C and constant darkness for four days. After this period mortality of mites was recorded using a dissection microscope Technival 2 (Carl Zeiss, Jena, Germany) at a magnification 25× and 40×. Mycosis on cadavers was documented by Olympus SZX12 equipped with an Olympus E-3 digital camera (Olympus Optical Co., Ltd., Tokyo, Japan) at 50× magnification. Each strain bioassay was conducted in 3 replicates, i.e. 3 times one plate with 96 mites tested.
Dose-response of R. robini to EPFs was assessed in three selected strains of Metarhizium spp. which showed high efficacy in previous single-dose experiments. The mean and the median time to death (LT50, the number of days until 50% of mites were dead) and lethal concentrations (LC50 and LC90) of conidia were estimated from cumulative mortality of mites at five concentrations ranging from 1×103 to 1×107 spores/ml of suspension. The bioassays were performed as described above except that mortality was checked daily. The control was treated with 0.05% Tween 80® solution. Each concentration test was repeated twice; 96 mites were used per replication.
Scanning electron microscopy of pathogenesis
Pathogenesis was studied in two selected EPF strains, BEA 02 and MET 08. Samples of R. robini females treated by either strain were collected at 24, 48, 72 and 96 hours after the application of fungal suspension. Mites were fixed and dehydrated in vapors from crystals of osmium tetroxide in Petri dishes properly sealed with Parafilm® at -20 °C in a freezer. After three weeks the Petri dishes with mites were placed in the fume hood and kept open for 24 hours to evaporate remaining osmium tetroxide. The following day the mites were mounted on aluminium stubs using a double-sided carbon tape and coated with gold using a Sputter Coater (Baltec-SCD 050). The mites were examined in the scanning electron microscope JEOL 7401-FE (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 4 kV.
Statistical analysis
Mortality in R. robini females was expressed as mean percentage ± standard error of the mean. A generalized linear model with a binomial distribution and logit link was used to analyse data. Treatment and replication were set as fixed effects. The analysis was performed in SAS® Studio for Linux (SAS Institute Inc. 2018) using the GLM procedure (PROC GENMOD) of SAS/STAT module (SAS Institute Inc. 2017). Means were separated by the least-square means (LSMEANS) statement of SAS with Tukey-Kramer adjustment for multiple comparisons. Dose-response experiment data were first subjected to survival analysis. The Kaplan–Meier product limit estimate calculated in the LIFETEST procedure in SAS/STAT module was used to determine both the mean and the median time to death (LT50) for each selected strain. Wilcoxon and log-rank test statistics (PROC LIFETEST) were used to test the global hypothesis that mortality (time to death) differed between strains. Data were further analysed using Probit analysis (PROC PROBIT) to estimate lethal concentrations (LC50 and LC90). In all tests P values <0.05 were considered statistically significant.
Results
In total, 17 strains of EPFs were isolated and identified as species B. bassiana, Beauveria brongniartii (Sacc.) Petch, I. fumosorosea, M. anisopliae and Metarhizium indigoticum (Kobayasi & Shimizu) Kepler, S.A. Rehner & Humber (Table 1). The results of bioassays revealed that while in the control treatment mortality of R. robini females was only 2.8% without any evidence of fungal infection, some strains belonging to genus Metarhizium were able to kill almost all treated mites within four days (Fig. 1). Mycosis on the cadavers followed by sporulation was observed in few mites treated by Beauveria spp. strains (Fig. 2 A) and almost on all cadavers of mites treated by Metarhizium spp. strains (Fig. 2 B, C). No obvious symptoms of mycosis were found four days after the treatment by strains of other EPF species. Observation of pathogenesis revealed that conidia germinated in 24 hours and in 48 hours were able to form appressoria (Fig. 3 A1-2). Strains of Metarhizium genera were the fastest in development of conidiophores and sporulation was observed as early as 72 hours after fungus application (Fig. 3 B3).
TABLE 1.
Strains of entomopatogenic fungi isolated within this study and used in bioassays.
FIGURE 1.
Mortality of Rhizoglyphus robini adult females treated with various strains of entomopathogenic fungi. See Table 1 for the key to EPF strains. Data presented are means (± SE), with three replicates of 96 mites for each strain. A generalized linear model was fitted and pairwise between treatment differences were tested using the least-square means. Different letters indicate significant differences between columns (P<0.05).
FIGURE 2.
Photographs (dissection microscope, 50×) of sporulated entomopathogenic fungi on Rhizoglyphus robini cadavers. A: Beauveria bassiana strain BEA 02, B: Metarhizium indigoticum strain MET 01, C: Metarhizium anisopliae strain MET 08.
The most virulent were particularly M. anisopliae strains MET 08 and MET 07 from the Czech Republic and M. indigoticum strain MET 01 from Israel causing mean mortality 99.3, 98.6 and 98.3%, respectively. The strain F52 caused also high mortality of R. robini females (80.6%). Other species of EPF turned out to be much less virulent against R. robini. The reference strain of I. fumosorosea CCM 8367 caused mortality less than 40%. Strains of the genus Beauveria showed very low acaropathogenic effect against R. robini females. Mortality ranged from 4.9% (BEA 04) to 25.0% (BEA 02). The effect of strain on virulence against R. robini was highly significant (χ2=4086.32, df=20, P<0.001). No significant differences were found among replications (χ2=1.41, df=2, P=0.493).
FIGURE 3.
Scanning electron micrographs showing pathogenesis of Beauveria bassiana strain BEA 02 (A) and Metarhizium anisopliae strain MET 08 (B) on Rhizoglyphus robini females at 24 (1), 48 (2), 72 (3) and 96 (4) hours after fungus application. A1: germinating conidia; B1: attachment of conidia to mite cuticule; A2: appressorium (ap) formation; B2 and A3: mycelium on the edge of the anal opening; B3: the beginning of sporulation; A4 and B4: details of conidiophores (cf) with conidia (c).
Cumulative mortality at the end of dose-response experiments reached 97.9, 99.0 and 99.5% in mites treated with the highest concentration of MET 01, MET 07 and MET 08 strain, respectively (Fig. 4). First mycosis was observed on the 3rd day after fungus application at the highest concentration when 22.3%, 12.0% and 14.0% of cadavers had symptoms of mycosis in MET 01, MET 07 and MET 08 strain, respectively. Four days after the treatment, mycosed cadavers were found also at concentrations 1×105 and 1×106 spores/ml (Fig. 5). Survival analysis of obtained data revealed no statistically significant effect of strain at the lowest concentration, i.e. 1×103 spores/ml (Wilcoxon test, χ2=1.705, P=0.426; log-rank test, χ2 = 1.654, P = 0.437) but highly significant differences were found at all higher concentrations (Wilcoxon test and log-rank test, P<0.001). The shortest median survival time (LT50=2.0 days) was estimated in strain MET 07 applied at concentration 1×107 spores/ml (Table 2). The log-probit regression lines describing relationship between concentration of MET01, MET07 and MET08 strains and mortality of R. robini (Fig. 6) have a form y = -3.830 + 0.957x, y = -3.315 + 0.617x and y = -3.324 + 0.711x, respectively. The estimated values of LC50 and LC90 were lowest in strain MET 01 (Table 3).
TABLE 2.
Corrected mortality, mean survival time (± SE) and median lethal time (LT50) of Rhizoglyphus robini adult females treated by suspensions of selected strains of Metarhizium spp.
Discussion
Fungal insect pathogens are important natural control agents for many insect and other arthropod pests. These pathogens have a potential to significantly reduce host insect populations (Burges 1981; Carruthers & Soper 1987; McCoy et al. 1988). Many EPF can be not only entomopathogenic but also acaropathogenic. The study by Chandler et al. (2000) describes many EPF species capable of attacking mites. In theory, Acari make good hosts for fungal pathogens because they are generally soft bodied and many inhabit environments with humid microclimates (Ferro & Southwick 1984) which favour infection and disease transmission (Hajek & St Leger 1994).
There are many studies that have focused on the use of the EPF H. thompsonii against mites. In laboratory experiments, Gerson et al. (1979) observed 94% mycosis of the carmine spider mite, Tetranychus cinnabarinus Boisduval (Acari: Tetranychidae), within four days of inoculation at a constant 100% relative humidity (RH), 68% mycosis at 100% RH for 18 h per day, and 23% mycosis at 100% RH for 6 h per day. Hirsutella thompsonii was also shown to be effective against the twospotted spider mite, Tetranychus urticae Koch in laboratory bioassays, causing 96.5% mortality with unformulated conidia and up to 99% mortality with the formulated Mycar® (Gardner et al. 1982). McCoy et al. (1971) applied the mycelia of H. thompsonii against the citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyiidae) infestation on citrus trees, observing sporulation after about 48 h, followed by a decline in mite infestation, which only began to recover 10–14 weeks later. In contrast, Sztejnberg et al. (1997) reported a failure of an isolate of H. kirchneri, obtained from the cereal rust mite (original accession number CMI 257456) to infect R. robini. Another EPF that has an acaropathogenic effect is Neozygites floridana (Weiser & Muma) Remaud. & S. Keller. It was first described as the cause of population declines in the Texas citrus mite, Eutetranychus banksi (McGregor) in Florida (Weiser & Muma 1966). Neozygites floridana is highly effective against the two-spotted spider mite, T. urticae and the tobacco spider mite, Tetranychus evansi Baker and Pritchard in several major crops where it causes natural epizootics (Humber et al. 1981; Klubertanz et al. 1991; Nordengen & Klingen 2006; Duarte et al. 2009).
TABLE 3.
Lethal concentrations of selected strains of Metarhizium spp. against Rhizoglyphus robini adult females.
In the present study, new strains of “nonspecialist” mitosporic fungi Beauveria spp., Isaria sp. and Metarhizium spp. were tested against the bulb mite R. robini under laboratory conditions. The most effective strains were those belonging to the genus Metarhizium. This genus is one of the most widely used fungus in mycoinsecticides throughout the world, mainly as an inundative control agent (Zimmermann 2007). According to Goettel et al. (1990) the host range of M. anisopliae includes Symphyla, Orthoptera, Dermaptera, Isoptera, Homoptera, Heteroptera, Diptera, Coleoptera, Hymenoptera, Siphonaptera, Lepidoptera and Acari, as well as some nontarget hosts, e.g. those belonging to Malacostrata (Amphipoda) and Ephemeroptera. A summary of the safety of M. anisopliae has been reviewed by Zimmermann (2007).
Metarhizium genus shows very high virulence to many mite species, as evidenced by several studies (Kaaya et al. 1996; Smith et al. 2000; Zimmermann 2007; Tomer et al. 2018). The results of the present study revealed that Czech strains of M. anisopliae MET 07 and MET 08 caused almost 100% mortality in R. robini females in four days of incubation. The highest virulence was found in M. indigoticum strain MET 01 from Israel in which the lowest median lethal concentration (LC50=1.01×104) was estimated compared to the above mentioned M. anisopliae strains. Their LC50 values were still much lower (4.74×104 and 2.36×105) than that reported by Wekesa et al. (2005) for M. anisopliae strain in which the lowest LC50 was 0.7×107 conidia/ml 7 days after application on T. evansi under laboratory conditions. The differences in efficacy may be due to different EPF strain or host species or due to the fact that in the present study conidia in suspensions were activated for 12 hours prior to their application which is known to synchronize germination of spores (Dillon & Charnley 1985, 1990). Recent study by Ment et al. (2020) demonstrated high efficacy against R. robini in M. brunneum isolate Mb7. Conidia of this fungus applied in-vitro at concetration 1×107 caused mortality of mites 43% and 100% at three and seven days post inoculation, respectively and the estimated LT50 value was 4.3 days. Drench application in potted onion experiments also significanly reduced bulb mite population compared to untreated control (Ment et al. 2020). Reference strain F52 of M. brunneum, the active ingredient of mycopesticide Met52® EC, is effective against many arthropod pests including the larvae of the black vine weevil Otiorhynchus sulcatus (Moorhouse et al. 1993; Bruck & Donahue 2007; Ansari & Butt 2013), chilli thrips Scirtothrips dorsalis (Arthurs et al. 2013), Japanese beetle larvae Popillia japonica (Behle et al. 2015; Krueger et al. 1992), the Asian longhorned beetle Anoplophora glabripennis (Gardescu et al. 2017; Clifton et al. 2020) and the tick Ixodes scapularis (Bharadwaj & Stafford 2010; Stafford & Allan 2010). In the present study F52 strain also caused high mortality of R. robini (80%) but lower than other Metarhizium spp. strains tested.
FIGURE 4.
Cumulative mortality of Rhizoglyphus robini treated by Metarhizium indigoticum strain MET 01 (A) and Metarhizium anisopliae strains MET 07 (B) and MET 08 (C). Data presented are means (± SE), with two replicates of 96 mites for each strain and concentration.
FIGURE 5.
Percentage of mycosed cadavers of Rhizoglyphus robini adult females treated by Metarhizium indigoticum strain MET 01 and Metarhizium anisopliae strains MET 07 and MET 08. Data presented are means (± SE), with two replicates of 96 mites for each strain and concentration.
The strains of other EPF species, Isaria sp. and Beauveria spp. tested in the present study showed much lower efficacy against R. robini than strains of Metathizium spp. Reference strain CCM 8367 of I. fumosorosea caused 40% mortality which was slightly higher compared to the two other I. fumosorosea strains tested. This strain was reported to be a promising biocontrol agent against several insect pests (Hussein et al. 2013, 2016; Prenerová et al. 2013) although recent findings demonstrated that some pests might be resistant to infection (Zemek et al. 2020). The strain also turned out to be virulent against T. urticae (Zemek et al. 2016). High virulence of I. fumosorosea against T. urticae was found earlier by Kim et al. (2008). This EPF species was found to be effective also against the broad mite, Polyphagotarsonemus latus (Bank) (Acari: Tarsonemidae) (Pena et al. 1996), T. cinnabarinus (Shi & Feng 2004a,b), Eutetranychus orientalis (Klein) (El-Sharabasy 2015) and the European red mite, Panonychus ulmi (Koch) (Graeff et al. 2017) whereas it provided only moderate effect against P. oleivora compared to B. bassiana and M. anisopliae (Robles-Acosta et al. 2019). When I. fumosorosea is applied together with other biological control agents, its side effects against non-target species like predatory mites or parasitoids needs to be assessed (Zemek et al. 2017). For example, in a recent study by Chen et al. (2020), the fungal entomopathogen I. fumosorosea exhibited low toxicity to the predatory mite Neoseiulus cucumeris (Oudemans).
High efficacy of B. bassiana against mite pests has been documented by several authors. Khoury et al. (2020) compared the virulence of blastospores and aerial conidia of different strains of B. bassiana against different life stages of T. urticae under laboratory and greenhouse conditions with high mortality after exposure mainly to blastospores. Under laboratory conditions, the LT50 values of conidia of the Lebanese strain of B. bassiana (concentration 1×107) was estimated to be 5.6 and 7.5 days for adults and for motile juveniles, respectively. Shi et al. (2008) demonstrated that B. bassiana had a significant ovicidal effect on the two-spotted spider mite, with up to 87.5% egg mortality in laboratory bioassays while a significant negative effect on reproductive potential of T. urticae females was reported by Shi and Feng (2009). Chandler et al. (2005), in a greenhouse experiment, demonstrated up to 97% reduction in T. urticae abundance when the commercial biopreparate Naturalis-L based on B. bassiana was applied. Wekesa et al. (2005) studied the pathogenicity of B. bassiana against T. evansi and determined the LC50 of 1.1×107 conidia/ml. Studies with other mite species reported promising results for B. bassiana against P. latus in laboratory bioassays and greenhouse trials (Peña et al. 1996) and with various fungal isolates against the false spider mite, Brevipalpus phoenicis (Geijskes) (Acari: Tenuipalpidae) under laboratory conditions (Rossi-Zalaf & Alves 2006). The strain GHA has been reported to be highly efficient in control of many insect pest species (Liu & Bauer 2008; Mukawa et al. 2011; Clavet et al. 2013; Parker et al. 2015) and was found to be virulent also against T. urticae (Ullah & Lim 2015). In the present study, however, the efficacies of all tested Beauveria spp. strains against R. robini were rather low confirming the fact that the virulence of infective propagules of B. bassiana may vary with respect to the arthropod host or their developmental stage (Khoury et al. 2020).
FIGURE 6.
Log-probit regression lines of concentration-mortality response of Rhizoglyphus robini to Metarhizium indigoticum strain MET 01 (A) and Metarhizium anisopliae strains MET 07 (B) and MET 08 (C).
The above results indicate that R. robini is more resistant to some EPF species than to others. This might be linked to some compounds found in R. robini which have been shown to possess antifungal activity. Leal et al. (1990a) described hexyl rhizoglyphinate and showed that it inhibited mycelial growth of several species of fungi. Further studies are needed to elucidate if this inhibitory effect is species-specific, e.g. if it negatively affects Beauveria spp. or Isaria sp. more than Metarhizium spp. The role of other compounds, such as the monoterpenoids robinal (Leal et al. 1990b) and isorobinal (Sakata et al. 1996) in adaptation of bulb mites to live next to some acaro/entomopathogenic fungi in soil environments also remains to be explored.
Conclusions
Acaropathogenic status was demonstrated in most strains of EPF isolated from soil of onion and garlic fields. The most virulent strains of M. anisopliae (MET 08) from the Czech Republic and M. indigoticum (MET 01) from Israel caused almost 100% mortality in R. robini females. Results of the present study thus indicate that particularly that species of EPF belonging to the genus Metarhizium can be promising new biocontrol agents against R. robini. Further research needs to be carried out to verify if application of EPF is viable as an alternative method to chemical control of bulb mites under greenhouse and field conditions.
Acknowledgements
This research was conducted with institutional support RVO:60077344 and co-financed by Grant Agency of the University of South Bohemia (project No. 018/2018/Z). It was also an integral part of a joint Czech and Israeli project titled ‘New biorational methods applied to control selected pests as an alternative to chemical pesticides to prevent contamination of soil and water resource’, supported by the Ministry of Education, Youth and Sports of the Czech Republic (project No. 8G15006) and the Ministry of Science, Technology and Space, State Israel (project No. 3-13035). We also acknowledge the Laboratory of Electron Microscopy facility at the Biology Centre CAS supported by the MEYS CR (LM2018129 Czech-BioImaging) and ERDF (No. CZ.02.1.01/0.0/0.0/16_013/0001775). The authors thank Dr. Jana Nebesářová and Ms. Martina Tesařová for their valuable advice on SEM imaging and Mrs. Olga Divišová and Mrs. Barbora Kozelková for their technical help.
