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. 2022 Jul 3;22(4):4. doi: 10.1093/jisesa/ieac040

Infectivity of Entomopathogenic Fungal Isolates Against Tarnished Plant Bug Lygus lineolaris (Hemiptera: Miridae)

Nguya K Maniania 1, Maribel M Portilla 2,✉, Fayaz M Amnulla 3, David K Mfuti 4, Andrei Darie 5, Geetika Dhiman 6, Ishtiaq M Rao 7
Editor: Kris Godfrey
PMCID: PMC9250697  PMID: 35780385

Abstract

Twelve isolates of entomopathogenic fungi belonging to Metarhizium robertsii, M. pinghaense, M. brunneum, Beauveria bassiana, and Isaria fumosorosea were screened against tarnished plant bug. All isolates were pathogenic, causing mortality from 28.8 ± 3.4 to 96.3 ± 2.7%. The LT50 values ranged from 2.7 to 6.0 d while the LT90 values varied between 6.6 and 15.0 d. Metarhizium robertsii isolate CPD6 (will be under the trade name NoVil) was among the isolates that caused high mortality within shorter times and was selected for study on developmental stages and greenhouse trial. The third-, fourth-, and fifth-instar nymphs, and adults were inoculated with 106, 107, and 108 conidia per ml of NoVil. All the stages were susceptible to fungal infection. However, third and fourth instars were the most susceptible with no significant differences in mortality across the three concentrations. On the other hand, mortality was dose-dependent with fifth-instar nymph and adult stages. The LT50 and LT90 values were also dose-dependent, with higher concentrations having shorter lethal-time values as compared to the lower concentrations. In the greenhouse, pepper plants were sprayed with NoVil and chemical insecticide Flonicamid (as industrial standard), before releasing adult tarnished plant bug. Mortality of 37.3, 75.5, and 76.3% was recorded in the control, NoVil, and Flonicamid, respectively. This study has identified NoVil as a potential mycoinsecticide candidate for the control of tarnished plant bug under greenhouse conditions. Further field testing on juvenile and adults is needed to evaluate the potential for in-field control.

Keywords: Beauveria bassiana, Metarhizium, biological control, NoVil, greenhouse

The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is the most common phytophagous species of the genus Lygus in North America (Broadbent et al. 2013). It causes damage to vegetables, fruits, greenhouse crops, canola, Brassica napus (L.), and B. rapa (L.) (Brassicales: Brassicaceae), and legume crops (Broadbent et al. 2002). UMass Extension Greenhouse Crops and Floriculture Program (2011) reported that lygus bugs feed in many Asteraceae flowers including dahlia Dahlia pinnata (Cav), chrysanthemums Chrysanthemus indicum (L.), sunflower (Helianthus spp.), daisy (Bellis spp.), calendula (Calendula spp.), and many other. Symptoms of feeding flowers can result in multiple shoots (stink marks), distortion and shedding of flowers buds. In Ontario, Canada, tarnished plant bug commonly infests greenhouse pepper Capsicum annuum (L.) (Solanales: Solanaceae) and cucumbers Cucumis sativus (L.) (Cucurbitales: Cucurbitaceae) (OMAFRA 2021). Pepper and cucumber damage in greenhouse conditions include destruction of growing points of young seedlings, distortion of flowers and young fruits. Fernandez et al. (2020) mentioned that in 2018, 17.5 million m2 of vegetables were grown in greenhouses in Canada, where pepper represented the second largest vegetable crop grown in this environment with 5.6 million m2.

Control of tarnished plant bug has mainly relied on the use of synthetic chemical insecticides, but in Ontario, Canada, only Beleaf 50 (Flonicamid) is registered for use in greenhouses (OMFRA 2021). Toxicity of these chemical insecticides, however, is a concern to nontarget and beneficial arthropods and safety to humans. Furthermore, tarnished plant bug has developed resistance to some insecticides in field populations (Snodgrass 1996, Snodgrass et al. 2009).

A major transformation in pest management philosophy and approach has recently been observed in Canada. Brownbridge and Buitenhuis (2019) documented that over 70% of Canadian floriculture greenhouse growers use biocontrol, which is playing a critical supporting role in biologically based integrated pest management strategies against a range of challenging pests including tarnished plant bug. Portilla et al. (2017) noted that since the late 1990s, there has been keen interest in decreased reliance on chemical insecticides for control of the tarnished plant bug, including efforts to find naturally occurring microbial pathogens that can be used for biological control.

Tarnished plant bug has been reported to be susceptible to entomopathogenic fungi (Bidochka et al. 1993; Steinkraus and Tugwell 1996; Liu et al. 2002, 2003; McGuire 2002; Leland and Snodgrass 2004; Al-mazra’awi et al. 2005; Leland et al. 2005; Leland and McGuire 2006; Sabbahi et al. 2007; Ugine 2011; Portilla et al. 2014, 2017, 2019). With exception to the study of Liu et al. (2002) that investigated the pathogenicity of a Metarhizium anisopliae (Metschn.) Sorok. isolate and other entomopathogenic fungi against tarnished plant bug, all the studies have been carried out with Beauveria bassiana isolates. This may be explained by natural occurrence of B. bassiana in Lygus spp. populations (McGuire 2002, Leland and Snodgrass 2004). Although M. anisopliae complex species has not been associated with tarnished plant bug in nature, its virulence can be evaluated under controlled experimental conditions (Hall and Papierok 1982).

The developmental stages of a host are among the biotic factors that can affect fungal infection (Ferron 1985) and has been reported to vary according to the stage. For example, in greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) and large pine weevil Hylobius abietis (L.) (Coleoptera: Curculionidae), their early stages have been reported to be more susceptible than older stages (Fransen et al. 1987 and Ansari and Butt 2012, respectively). However, in the case of legume flower thrips, Megalurothrips sjostedti (Trybon) (Thysanoptera: Thripidae) adult stages have been found to be more susceptible to fungal infection than the larval and pupal stages (Ekesi and Maniania 2000). The same is true for the nypmphal stage in the case of brown planthopper Nilaparvata lugens (Stal) (Hemiptera: Delphacidae) and whitebacked planthopper, Sogatella furcifera Horvath (Hemiptera: Delphacidae) (Geng and Zhang 2008). Since crops are often infested with several different stages of the pest, understanding which stage is most susceptible to infection is important for the development of management tactics (Ferron 1985). Although nymphal and adult stages of tarnished plant bug have been reported to be susceptible to fungal infection, only one study that has investigated the susceptibility of different developmental stages to B. bassiana infection (Portilla et al. 2014), but no published information is available for the susceptibility of tarnished plant bug stages to M. robertsii. Therefore, the present study investigates the pathogenicity of entomopathogenic fungal isolates including M. robertsii strain CPD6 (to be marketed as NoVil) (Maniania et al. 2020) against tarnished plant bug. The most virulent isolate(s) selected and the susceptibility to different developmental stages of tarnished plant bug to these isolates were compared. In addition, its performance was evaluated under greenhouse conditions.

Materials and Methods

Insects

Insects were obtained from USDA/ARS Southern Insect Management Research Unit, Stoneville, MS. They were reared according to the technique described by Portilla et al. (2011), which uses a semisolid artificial diet and allows for the mass production of even-aged individuals. Insects were held under 12:12 (L:D) h photoperiod, 27°C, and 50–70% RH.

Fungal Isolates

Twelve fungal isolates originating from soil using ‘Galleria bait method’ belonging to M. robertsii (six isolates) including NoVil strain, M. pinghaense (two isolates), M. brunneum (one isolate), B. bassiana s.l. (two isolates), and Isaria fumosorosea (one isolate) were screened against adult tarnished plant bug (Table 1). They were obtained from the culture collection at Crop Defenders Ltd, Ontario, Canada. Fungal isolates were cultured on Sabouraud dextrose agar medium supplemented with 0.5% yeast extract and maintained at room temperature (24–28°C). Conidia were harvested from 2- to 3-wk-old sporulating cultures (>95% viability for all isolates) and suspended in 10 ml 0.05% Silwet 408 (www.momentive.com) in universal bottles containing glass beads (3 mm). The suspension was vortexed for 5 min at 100 rpm to homogenize suspension. Conidial concentration was determined using hemocytometer. Different concentrations for the most virulent strain (NoVil) were obtained through serial dilutions (106, 107, and 108 conidia per ml).

Table 1.

Origin of fungal isolates tested against adult Lygus lineolaris

Fungal species Accession number Source of origin
Metarhizium robertsii CPD1 Soil1
CPD2 Soil1
CPD62 Soil1
CPD20 Soil1
CPD30 Soil1
CPD31 Soil1
M. pinghaense CPD10 Soil1
CPD24 Soil1
M. brunneum CPD37 Soil1
Beauveria bassiana CPD28 Soil1
CPD61 Endophyte from cucumber leaf
Isaria fumosorosea Soil1
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‘Galleria bait method’ was used for isolation.

Strain under development as a commercial mycoinsecticide with the name of NoVil.

Bioassays

Screening

Adults of tarnished plant bug were used in this experiment. Insects were exposed to a standard concentration of 1 × 108 conidia per ml for each isolate by immersing them in a fungal suspension for 5 s. In the control, insects were immersed in 0.05% Silwet 408 solution for 5 s. Test insects were then transferred to 9.5-cm aerated plates whose bottom was lined with filter paper and 3.5-cm parafilm bags containing nonautoclavable diet that served as food source. Mortality was recorded from day 3 to day 7 postinoculation. Dead tarnished plant bug adults were retained in the same plate until completion of the 7-d trial to observe sporulation (presence of mycelial growth). Treatments were randomized and consisted of 15–20 insects per replicate each and replicated four times.

Susceptibility of Different Developmental Stages to Fungal Infection

Adults and third-, fourth-, fifth-instar nymphs of tarnished plant bug were used in this study. Metarhizium robertsii isolate NoVil which was among the fungal isolates that caused high mortality and short LT50 and LT90 values during screening bioassays was selected for this study. Test insects were exposed to three different fungal concentrations (106, 107, and 108 conidia per ml) using the immersing method described above. Mortality was recorded from day 3 to day 7 postinoculation. No molting was recorded. Treatments were randomized and consisted of 15–20 insects per replicate each and replicated four times.

Greenhouse Trial

Pepper Plants

Five-month-old pepper plants ( ‘Bell Boy’ variety) were used. They were planted on rockwool slabs (IP 20/75) and then transferred to 15-liters plastic pots filled with BM 6 soil All Purpose (www.berger.ca). Nutrients (1 liter fertilizer was uniformly distributed to 24 pots of pepper plants): CaNO3 (22%), iron chelate 13% (0.5%), NO3 (5%), monopotassium phosphate (6%), MgSO4 (0.6%), MgNO3 (10%), micronutrient mix (0.5%) were dispensed daily by hand. Individual pepper plants were covered with netting (60 cm diameter × 150 cm height). The top of the plastic pot was lined with netting to catch dead insects that fell from the plant. The netting did not interfere with nutrient dispensing.

Treatments

Treatments consisted of 1) control, 2) M. robertsii strain NoVil, and 3) Beleaf 50 SG insecticide (Flonicamid) [N-(cyanomethyl)-4-(trifluoromethyl) pyridine-3-carboxamide] (ISK Biosciences Corporation, Auburn Road, Concord, OH). For the control was used a water solution of canola oil (0.36%) and Silwet 408 (0.02%) (360 ml of canola oil and 20 ml of Silwet 408 was added to 620 ml of water), the strain NoVil treatment was formulated conidia in canola oil and Silwet 408 with the same ration as control with a concentration of 107 conidia per ml, the Fonicamid nicotinamide treatment, was used according to labeled instructions with a recommended dose of 0.3 g/liter. Flomicamid affects insects by contact causing irreversible feeding cessation. Spray applications were carried out using 1-liter spray bottles. Plants were sprayed (10 ml per plant/treatment) first before the release of the insects. Twenty 2-d-old tarnished plant bug adults (unknown sex) were released per plant/cage 1 h after sprayed or until treatments were air-dry. Treatments consisted of two plants each and replicated four times in a randomized block design. Dead insects were removed daily. The experiment ran for 10 d.

Statistical Analysis

Percent mortalities were corrected for control mortality using Abbott’s formula (Abbott 1925) and arcsine-transformed to normalize the data before analysis of variance. Differences between means were compared using Tukey HSD test and P = 0.05 was considered as significant. Lethal time to 50% and 90% mortality (LT50 and LT90), lethal concentration to 50% and 90% mortality (LC50 and LC90) values were estimated by Probit analysis. All data analyses were performed using R version 3.4.3 statistical software package (R Core Development Team 2019).

Results

Infectivity of Entomopathogenic Fungal Isolates Toward Tarnished Plant Bug Adults

Mortality in the control was 32.4 ± 3.5%. All the fungal isolates tested except for I. fumosorosea were pathogenic to adult tarnished plant bug. Mortality varied significantly (F12, 40 = 13.02; P < 0.001) between fungal isolates with I. fumosorosea having the lowest mortality rate of 28.8 ± 3.4% and M. robertsii isolate NoVil and M. brunneum having the highest mortalities of 95.0 ± 3.3% and 96.3 ± 2.7%, respectively (Table 2). The LT50 values ranged from 2.7 to 6.0 d with M. robertsii isolate CPD30 and B. bassiana isolate CPD61 having the shortest LT50 and M. robertsii isolate CPD1 and M. pinghaense CPD24 the longest (Table 2). The LT90 values ranged from 6.6 d with M. robertsii NoVil and B. bassiana CPD28 to 15.0 d with M. pinghaense CPD24. Since NoVil strain was among the fungal isolates that caused high mortality within the shortest time, it was selected for the study on the susceptibility of different developmental stages to fungal infection.

Table 2.

Pathogenicity of fungal isolates applied at a concentration of 108 conidia per ml to adult Lygus lineolaris at 7 d postinoculation

Fungal species Isolate % Mortality (mean ± SE) LT50 (d) (95% fiducial limit) LT90 (d) (95% fiducial limit) Intercept
Control 32.4 ± 3.5d — —
Metarhizium robertsii CPD1 62.5 ± 5.4bc 6.0 (5.8–6.2) 9.1 (8.7–9.7) −2.40 ± 0.03
CPD2 81.8 ± 3.3abc 3.9 (3.3–4.3) 10.9 (9.7–12.8) −0.71 ± 0.03
CPD61 95.0 ± 3.3a 3.5 (3.3–3.6) 6.6 (6.4–6.8) −1.44 ± 0.01
CPD20 82.5 ± 3.5abc 4.8 (4.6–5.0) 8.9 (8.4–9.6) −1.47 ± 0.03
CPD30 92.0 ± 2.3ab 2.7 (2.2–3.1) 7.4 (7.0–8.0) −0.74 ± 0.03
CPD31 94.05 ± 3.3a 4.2 (4.0–4.3) 6.9 (6.7–7.2) −1.93 ± 0.03
M. pinghaense CPD10 85.8 ± 3.1abc 4.5 (4.3–4.6) 8.0 (7.6–8.5) −1.55 ± 0.03
CPD24 60.5 ± 4.9c 6.0 (5.6–6.7) 15.0 (12.7–19.2) −0.86 ± 0.02
M. brunneum CPD37 96.3 ± 2.2a 4.3 (4.1–4.4) 7.1 (6.8–7.4) −1.91 ± 0.03
Beauveria bassiana CPD28 95.0 ± 1.2ab 4.1 (4.0–4.2) 6.6 (6.4–6.8) −2.11 ± 0.03
CPD61 94.0 ± 2.9ab 2.8 (2.3–3.2) 7.8 (7.3–8.5) −0.74 ± 0.03
Isaria fumosorosea N/A 28.8 ± 3.4d — —
F 12, 40 = 13.02, P < 0.001
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Means (% ± SE) within column bearing the same letter are not significantly different by Tukey HSD (P = 0.05) test. (—) = not calculated.

Strain under development as a commercial mycoinsecticide with the name of NoVil.

Susceptibility of Developmental Stages of Tarnished Plant Bug to Fungal Infection by M. robertsii NoVil

All the insect developmental stages were susceptible to fungal infection at all the three concentrations (106, 107, and 108 conidia per ml) except for fifth instar and adult stage. For these two stages there were no significant differences observed at the lowest concentration (Table 3). Third- and fourth-instar nymphs were the most susceptible compared to fifth-instar nymphs and adults. No significant differences in mortality were observed between the concentrations: third-instar nymph (F = 1.00; df = 2, 11; P = 0.41) and fourth-instar nymph (F = 1.00; df = 2, 11; P = 0.41). Mortality was dose-dependent with fifth-instar nymph (F = 4.60; df = 2, 11; P < 0.05) and adult stage (F = 37.6; df = 2, 11; P < 0.001). At the lowest concentration (106 conidia per ml), mortality was 27.2 ± 3.9% and 31.2 ± 4.6%, respectively, with fifth instar and adult stage. At the highest concentration, mortality was 81.0 ± 3.9% and 98.1 ± 2.3% with fifth instar and adult, respectively (Table 3). The LT50 and LT90 values were dose-dependent, with higher concentration having shorter lethal-time values as compared to the lower concentrations (Table 3). Lethal concentration (LC50 and LC90) values varied according to the developmental stages, with third-instar nymph having the lowest values: 3.2 × 104 and 7.6 × 105 conidia per ml, respectively. The fourth-instar nymph LC50 and LC90 values followed with 1.9 × 105 and 1.3 × 106 conidia per ml, respectively (Table 4). On the other hand, fifth-instar nymphs had the highest LC50 (6.2 × 106 conidia per ml) and LC90 (3.3 × 108 conidia per ml) values. LC50 and LC90 values of adults were 2.5 × 106 and 3.2 × 107 conidia per ml, respectively (Table 4).

Table 3.

Susceptibility of the different developmental stages of Lygus lineolaris exposed to different concentrations of Metarhizium robertsii NoVil: mortality and lethal times at 7 d postinoculation

Developmental stages Treatment % Mortality (mean ± SE) LT50 (d) (95% fiducial limits) LT90 (d) (95% fiducial limits)
Third Control 37.0 ± 1.8b — —
106 91.5 ± 4.1a 3.5 (3.4–3.6) 4.8 (4.7–4.9)
107 100a 3.2 (3.1–3.3) 4.5 (4.3–4.6)
108 100a 2.6 (2.4–2.8) 3.8 (3.7–3.9)
F 2, 11 = 1.00; P = 0.41
Fourth Control 36.3 ± 2.4b — —
106 86.7 ± 5.2a 3.3 (3.2–3.4) 4.8 (4.6–5.0)
107 100a 3.4 (3.3–3.5) 4.4 (4.3–4.6)
108 100a 2.3 (1.9–2.5) 3.6 (3.4–3.7)
F 2, 11 = 1.00; P = 0.41
Fifth Control 32.5 ± 7.5b — —
106 27.2 ± 3.9b — —
107 57.3 ± 5.6ab 4.3 (4.2–4.4) —
108 81.0 ± 3.9a 3.5 (3.3–3.6) 5.9 (5.6–6.4)
F 2, 11 = 4.6, P < 0.05
Adult Control 40.0 ± 4.1c — —
106 31.2 ± 4.6c — —
107 77.5 ± 3.9b 5.5 (5.3–5.7) —
108 98.1 ± 2.3a 3.5 (3.4–3.7) 6.3 (6.1–6.4)
F 2, 11 = 37.6, P < 0.001
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Means (% ± SE) within column and within stages bearing the same letter are not significantly different by Tukey HSD (P = 0.05) test. (—) = not calculated.

Table 4.

Susceptibility of the different developmental stages of Lygus lineolaris exposed to different concentrations of Metarhizium robertsii NoVil: lethal concentrations

Developmental stage LC50 (95% fiducial limits) LC90 (95% fiducial limits) Intercept (± SE) P-values
Third 3.2 × 104 (1.0–19 × 104) 7.6 × 105 (0.4–17 × 1 05) −4.16 ± 2.26 0.066
Fourth 1.9 × 105 (0.1–4.4 × 105) 1.3 × 106 (0.7–2.7 × 106) −8.13 ± 3.51 0.021
Fifth 6.2 × 106 (3.7–9.9 × 106) 3.3 × 108 (1.4–3.0 × 108) −5.05 ± 0.68 0.0001
Adult 2.5 × 106 (1.7–3.6 × 106) 3.2 × 107 (1.9–6.0 × 107) −7.44 ± 0.85 0.0001
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Greenhouse Trial

Figure 1 illustrates mortality curves of tarnished plant bug following exposure to pepper plants previously treated with M. robertsii strain NoVil and chemical insecticide. Mortality analyzed by the test of equality over strata statement in Wilcoxon test (survival probability) PROC LIFETEST indicated high significant differences between treatments (χ2 = 10.19, df = 3, P = 0.0061). It is clear in Fig. 1 that the insecticide treatment plateaued faster than NoVil treatment; however, NoVil reached mortality as high as the insecticide treatment 10 d after exposure with no significant differences among those treatments (Fig. 2). Figure 2 shows that mortality at all evaluation times was lower in control than the other treatments. Observations of time to mortality was measured through routine posttreatment with high significant differences at 3 d (F = 28.99; df = 2, 3; P < 0.001), 5 d (F = 15.12; df = 2, 3; P < 0.002), and 10 d (F = 33.57; df = 2, 11; P < 0.001) after exposure (GLM procedure) (Fig. 2).

Fig. 1.

Fig. 1.

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Survival probability of tarnished plant bug adults, Lygus lineolaris following exposure to pepper plant foliage treated with Metarhizium robertsii CPD6 (NoVil) and the insecticide Flonicamid. Twenty insects per treatment were released after spray application of the treatments. LIFETEST of Equality Over Strata (P = 0.05).

Fig. 2.

Fig. 2.

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Cumulative mortality percentage of tarnished plant bug adults, Lygus lineolaris following exposure to pepper plant foliage treated with Metarhizium robertsii CPD6 (NoVil) and insecticide the Flonicamid. Twenty insects per treatment were released after spray application of the treatments. Bars within the group labeled with a different letter were significantly different at P = 0.05 (Tukey test).

Discussion

All the fungal species and isolates tested were pathogenic to adult tarnished plant bug except for I. fumosorosea. There was, however, variation in pathogenic activity between fungal isolates. Such variations among entomopathogenic fungal isolates have been reported for many insect pests (Ekesi and Maniania 2000), including tarnished plant bug. For instance, mortality caused by 32 isolates of M. anisopliae and B. bassiana against second-instar nymph tarnished plant bug was found to vary between 35 and 95% (Liu et al. 2002). In another study, Sabbahi et al. (2007) reported mortality that ranged from 23.3 to 100% of adult tarnished plant bug by 16 isolates of B. bassiana. Application of three isolates of B. bassiana caused mortality by mycosis between 33.0 and 80% in adults and for between 36 and 53% in nymphs of tarnished plant bug (Leland and McGuire 2006). The LT50 and LT90 values also varied according to fungal isolates as reported by other authors (Liu et al. 2002, Leland et al. 2005). Based on mortality and LT90 values, M. robertsii isolate NoVil was selected for further studies. The choice of M. robertsii NoVil over B. bassiana CPD28 that had the same LT90 values could be explained by the fact that NoVil has also been found to be virulent against other arthropod pests including pepper weevil, Anthonomus eugenii Cano and cranberry weevil, Anthonomus musculus Say (Coleoptera: Curculionidae), green peach aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and two-spotted red spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) (Maniania et al. 2020). Furthermore, this strain is under development as a commercial mycoinsecticide.

An isolate with broad-spectrum activity would be more desirable economically point of view than strict specificity isolate (Leland and McGuire 2006). Metarhizium complex species and B. bassiana are ubiquitous pathogens recorded on many hosts (Veen 1968); therefore, can be tested against insects that are not associated with them in nature and be developed as biopesticides. It is generally admitted that strains isolated from a particular host remain highly virulent to that host (Liu et al. 2002, Leland and McGuire 2006, Portilla et al. 2019). On the other hand, fungal strains isolated from a particular host have shown to be virulent to other hosts. For instance, Leland et al. (2005) reported that application of B. bassiana isolate grass hopper active, isolated from Chrysomelidae, caused higher mortality of tarnished plant bug in the field trials than B. bassiana isolates from tarnished plant bug and the western tarnished plan bug (Lygus hesperus Knight) (Hemiptera: Miridae). Similarly, Liu et al. (2002) reported that a strain of B. bassiana isolated from corn bug Eurygaster integriceps (Puton) (Hemiptera: Scutelleridae) and a strain of M. anisopliae isolated from honeycomb moth Galleria mellonella (L.) (Lepidoptera: Pyralidae) were found to be virulent against tarnished plant bug nymphs.

Third- and fourth-instar nymphs were more susceptible to NoVil than fifth-instar nymph and adult stages of tarnished plant bug as illustrated by lethal-time and lethal-dose mortality values (Tables 3 and 4). Similar results were reported by Meng et al. (2017) with another hemipteran, the litchi stinkbug Tessaratoma papillosa (Drury) (Hemiptera: Tessaratomidae). These authors observed that second-instar nymphs were more susceptible to B. bassiana infection than fifth-instar nymphs and adults mortality being 88.9, 75.6, and 65.6% with second- and fifth-instar nymphs and adults, respectively. The LT50 values were 4.3, 6.4, and 7.9 d with second- and fifth-instar nymphs and adults, respectively. The lethal concentrations were lower with the second-instar nymphs than the fifth-instar nymphal and adult stages, which is consistent with the present study. Portilla et al. (2014) reported less susceptibility on second than third, fourth, and fifth instars. They obtained 52.5% infection, that was significantly lower than that of the rest of the tarnished plant bug stages, and it took about two times longer for second instar for later instars and adults to die. However, similarly to our study, Portilla et al. (2014) found no significant differences in mortality between second instar and control.

In the preliminary greenhouse trial, no significant difference in mortality was observed between M. robertsii strain NoVil and the insecticide Flonicamid 10 d after treatment. As expected, mortality was initially low in the NoVil treatment, but increased through the time (Fig. 1). These results were comparable with those from previous laboratory studies that reported high mortality for Lygus spp. Beginning on day 3 increasing through time (Leland et al. 2005; Portilla et al. 2014, 2017). Entomopathogenic fungi are known to kill their hosts slowly and this may be influenced by many factors including biotic factors such as developmental stage of the host and concentration of the pathogen (Inglis et al. 2001). Adult and fifth-instar stages were susceptible to infection to NoVil only at the highest concentrations (Tables 3 and 4). In the greenhouse trial, insects were not directly sprayed with the inoculum but were exposed to treated foliage. The high mortality obtained in this investigation with NoVil demonstrated mortality that did not differed from an insecticide 10 d after treatment, clearly suggesting that the tarnished plant bug adults picked up spores as they walked on plants. These results agree with findings from Portilla et al. (2019) where mortality of tarnished plant bug and sporulation of B. bassiana by contact demonstrated the capacity of tarnished plant bug adult to acquire a lethal dose of conidia or propagate the fungus by walking or feeding on infected cotton terminals. However, it has also been shown that insects sprayed directly with a fungal suspension exhibited higher mortality than those expose by contact (Fernandez et al. 2001, Inglis et al. 2001, Portilla et al. 2019).

In conclusion, the present study has identified M. robertsii isolate NoVil as virulent against tarnished plant bug and should be considered as a candidate mycoinsecticide for tarnished plant bug. This study also demonstrated that younger stages are more susceptible to fungal infection than older stages. This is an important factor that should be considered when applying a mycoinsecticide in the field.

Acknowledgments

The project was cofunded by the Canadian Agricultural Partnership (CAP) through Ontario Greenhouse Vegetable Growers (OGVG). Authors would like to thank Tabatha Nelson, Essanya Winter, and Henry Winter ARS-USDA, Southern Insect Management Research Unit (SIMRU), Stoneville, MS, for maintaining the tarnished plant colonies.

Contributor Information

Nguya K Maniania, Crop Defenders Ltd, 3940 Highway 3, Maidstone, ON N0R 1K0, Canada.

Maribel M Portilla, USDA/ARS, Southern Insect Management Research Unit, Stoneville, MS 38776, USA.

Fayaz M Amnulla, Crop Defenders Ltd, 3940 Highway 3, Maidstone, ON N0R 1K0, Canada.

David K Mfuti, International Centre of Insect Physiology and Ecology, P.O. Box 30772-00100 Nairobi, Kenya.

Andrei Darie, Crop Defenders Ltd, 3940 Highway 3, Maidstone, ON N0R 1K0, Canada.

Geetika Dhiman, Crop Defenders Ltd, 3940 Highway 3, Maidstone, ON N0R 1K0, Canada.

Ishtiaq M Rao, Crop Defenders Ltd, 3940 Highway 3, Maidstone, ON N0R 1K0, Canada.

Author Contributions

N.K.M.: incepted and designed the experiments and wrote the original draft; M.P.: insect supply, finalized the writing and editing of the manuscript; F.M.A. and G.D.: performing the experiments in the laboratory and greenhouse and data collection; D.K.M.: application of statistical to analyze the data; A.D.: managed grant submission; I.M.R.: supervised and managed all financial concerns.

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