Microbial Control Of Spider Mites Based On Fungal Biocontrol Agents

Phytophagous spider mites, such as carmine spider mite Tetranychus cinnabarinus, are economically important pests infesting a wide range of plants grown in the field or greenhouse worldwide. Long-term chemical control of the mite pests has caused their high-level resistance to almost all acaricides due to the super life strategy of their small size, fast development and large fecundity. Thus, spider mite control encounters a great challenge and increasing interest turns to microbial control. Entomopathogenic fungi, such as Beauveria bassiana, Metarhizium anisopliae and Paecilomyces fumosoroseus, are important biocontrol agents that have been formulated against a variety of crop and forest insect pests. Such fungi may have potential for spider mite control although mites infrequently suffer from their infection in the field. However, there is a paucity of researches on the control of spider mites by the fungal agents. Technical difficulties with laboratory bioassay of fungal biocontrol agents on tiny mites hinder the progress of microbial control of spider mites.This study sought to develop an efficient bioassay system for evaluating the virulence of various fungal agents towards the eggs and adult females of T. cinnabarinus. A B. bassiana isolate (Bb2860) with high virulence was then chosen to test for its compatibility with ten acaricides, the lethal effect of its interaction with the sub-lethal rates of pyridaben on the mite eggs, and the influence of different temperature and humidity combinations on the ovicidal activity of its formulation. Finally, field trials of oil-based emulsifiable formulations of selected fungal isolates in conjunction with low application rates of selected acaricides were carried out for control of spider mites in citrus orchards of Zhejiang and in cotton fields of Xinjiang, respectively. The results are summarized as follows.Virulence of fungal pathogens to T. cinnabarinus eggs. Ten isolates of B. bassiana (Bb), M. anisopliae (Ma) and P. fumosoroseus (Pfr) were assayed for their virulence towards the eggs of T. cinnabarinus by developing a novel bioassay system. The eggs were naturally laid on detached fava bean leaves on agar in Petri dishes at the regime of 25℃and 12:12 L:D. Aqueous suspensions of aerial conidia of each isolate were sprayed onto the leaves, yielding three tier concentrations (no. conidia mm^-2) with each including 3 or 4 replicates (30-65 eggs per leaf or replicate). During a 12-day observation period after spray, the infected eggs shrunk in shape, then turned orange-brown for B. bassiana and P. fumosoroseus or dark-gray for M. anisopliae, and eventually had outgrowths of the sprayed fungus when maintained under moist conditions. Egg hatch rates at different concentrations increased with post-spray time but had no change from day 8 or 9 onward. The egg mortalities on day 9 differed significantly among the fungal species or isolates tested. Based on the LC50 estimates determined by the concentration-mortality relationships of all isolates from probit analysis, Bb2860 with an LC₅₀ of 548 conidia mm^-2 was highly virulence to the mite eggs, followed by Pfr116 (848 conidia mm^-2) and Pfr153 (913 conidia mm^-2). The LC₅₀s of two M. anisopliae isolates were 1717 and 1985 conidia mm^-2, showing less virulence. Other isolates had poor or very poor virulence to the mite eggs. Our results confirmed the ovicidal activity of the three fungal species, suggesting a feasibility to search for more ovicidal isolates from the fungal species that may serve as biocontrol agents of spider mites such as T. cinnabarinus.Virulence of fungal pathogens to female adults of T. cinnabarinus. Ten isolates of B. bassiana (Bb2860 and Bb734), P. fumosoroseus (Pfr116 and Pfr153) and M. anisoplae (Ma759, Ma456, Ma727, Ma5197, Ma576 and Ma2080) were assayed for their virulence against the female adults of T. cinnabarinus by developing a bioassay system different from the egg system. A pair of detached fava bean leaves sharing the petiole were prepared in advance to have hairy roots grown from the petiole into the 2% agar of glass bottle (2 cm in diameter, 4 cm in height) standing up in a Petri dish (9 cm diameter, fixed with 2% agar) and then was artificially infested with 10-15 female adults no more than 24h after last ecdysis. Adults on the paired leaves were separately exposed to sprays of conidial suspensions under a Potter Spray Tower at the concentrations of 29-43 (low), 195-260 (medium) and 1027-1946 (high) conidia mm^-2 for the Bb isolates, or at 39-78, 119-308 and 1528-1554 conidia mm^-2 for the Pfr isolates, or at 21-44, 130-238 and 740-1626 conidia mm^-2 for the Ma isolates. Each of the concentrations plus a black control (sprayed with 0.02% Tween 80 only) was replicated 6-8 times with each including 10-15 adults on a pair of leaves. After exposure, the detached leaves with mites were maintained at the regime of 25°C and 12:12 L:D for 9 days and examined daily for mortality records. The three concentrations resulted in the mortalities of 31.9-87.7% for B. bassiana, 19.4-77.7% for P. fumosoroseus and 24.2-80.3% for M. anisopliae, respectively. Observations from each assay fit very well to time-concentration-mortality model, and generated the estimates of LC50 varying with post-spray days and LT50 depending on conidial concentrations. Based on the fitted results, Bb2860 and Bb743 had high virulence to the mite adults due to small LC50s [177 and 42 conidia mm^-2 on day 9] and short LT50s (5.2 and 6.2 days at 750 conidia mm^-2). Pfr116 and Pfr153 showed moderate virulence (LC₅₀: 587 and 1240 conidia mm on day 9). Among the M. anisopliae isolates, Ma456 and Ma727 also showed high virulence (LC₅₀: 179 and 197 conidia mm^-2 on day 9; LT₅₀: 7.2 and 7.6 days at 750 conidia mm^-2). Other isolates except Ma2080 had the LC₅₀s of 424-632 conidia mm^-2 on day 9, falling in the median virulence group. The isolate Ma2080 with poor virulence caused a calibrated adult mortality of less than 50% even at the high concentration, making it impossible to reasonably estimate its LC₅₀ and LT₅₀. Thus, the isolates Bb2860, Bb734, Ma456 and Ma727 were desirable candidates against spider mites.Environmental effect on ovicidal activity of an emulsifiable formulation. Aerial conidia of Bb2860 in an oil-based emulsifiable formulation had germination rates of >95% after 24-h exposure to the combined regimes of 20, 25 and 30℃with 51, 74 and 95% RH although the first 12-h germination rates differed among the regimes. The formulation was assayed for its ovicidal activity towards T. cinnabarinus at the concentrations of 0 (control), 18, 160, and 693 conidia mm^-2 sprayed separately onto the mite eggs laid on fava bean leaves, followed by exposing the eggs to the combined regimes for hatch. Generally, hatched proportions increased over post-spray days and decreased with the elevated fungal rates and no more eggs hatched from day 9 or 10 onwards. The hatched proportions from all the regimes over post-spray days fit quite well (r²=0.88) to a logistic equation, in which temperature, humidity, and their interactions with or without the fungal spray rate were recognized affecting significantly the daily hatch rate. Counts of the hatched and unhatched eggs from the different regimes resulted in the estimates of final egg mortalities of 15.0-40.4% at the low fungal rate, 48.9-66.6% at the median, and 62.9-87.5% at the high, but only 5.6-11.3% in blank controls. Probit analyses of the egg mortalities versus the fungal rates generated the LC₅₀s of 65-320 conidia mm^-2 at all the regimes and of only 65-78 at 25-30℃with 74-95% RH. Thus, the emulsifiable formulation was highly capable of killing the mite eggs at the tested regimes and could be promising for spider mite control.Compatibility of acaricides with B. bassiana and fungus-pyridaben interaction on mite eggs. Ten chemical acaricides were tested for their compatibilities with the selected isolate Bb2860 by examining the germination rates of conidia in 24-h liquid cultures including three rates of each chemical. Pyridaben, hexythiazox and propargite were very compatible with the fungus even at their field application rates. Four bioassays were then performed with T. cinnabarinus eggs using the non-touch leaf method. The eggs laid on detached fava bean leaves were exposed to gradient conidial sprays of B. bassiana alone in Assay 1 or together with the pyridaben rates of 0.05, 1.0 and 2.5 AIμg ml^-1 in Assays 2-4, respectively. The percentages of hatched eggs decreased with the increasing conidial concentration and increased over post-spray days at a given concentration but had no more change from day 9 onwards. Final egg mortalities reached 65.4, 72.3, 77.7 and 85.3% at -1000 conidia mm^-2 in Assays 1-4. Ovicidal activity of B. bassiana [LC₅₀ with 95% CI: 546 (406-818) conidia mm^-2] was enhanced significantly by fungal sprays including the three rates of pyridaben [LC₅₀s: 322 (220-527), 199 (144-282), and 75 (48-103) conidia mm^-2]. The effects of both agents and their interactions on the daily hatch rate over post-spray days were illustrated well with the fitted time-concentration-hatch relationship. The results indicate an alternative to exploiting fungal/chemical interactions against spider mites.Field efficacy of emulsifiable B. bassiana formulation against citrus red mites. The emulsifiable formulation of Bb2860 conidia was applied alone or together with pyridaben 15% EC at～5% of its labeled application rate for control of citrus red mite Panonychus citri in three citrus orchards (Trials 1-3) of Huangyan, Zhejiang, southern China during the mild, rainy season of autumn 2005. Aqueous dilutions (150 L ha^-1) of two fungal rates (1.5 and 3.0×10¹³ conidia/ha in Trials 1 and 2; 1.2×10¹² and 1.2×10¹³ in Trial 3) or the low chemical rate (562.5 AI mg ha^-1) were sprayed to plots in randomized blocks (40-m-long row of trees per plot, 3 plots per treatment) with mean density of 17.8, 15.0 and 12.0 mites per leaf in Trials 1-3, respectively. Based on percent density declines and relative efficacies estimated by sampling all plots at 3-5 day intervals, the two fungal rates in each trial gave significant control and the fungal/chemical combinations always resulted in better control. The pyridaben-inclusive sprays of≥1.5×10¹³ conidia ha^-1 applied twice at 15-day interval provided excellent mite control for 35 days, yielding overall mean efficacies of 83.2-92.3% and mite density declines of 73.8-91.2%. In contrast, the relative efficacy in a standard pyridaben treatment (11.25 AI g ha^-1) reached 83.8% on day 3 but decreased from 72.0% on day 5 to 4.1% on day 15. Field conditions, featured with the records of～20°C and≥95% RH for 216 and 300 h in Trials 1 and 2 (35 days) and 52 h in Trial 3 (15 days), were favorable. The effects of local weather on the trials in topographically different hillside orchards are discussed. Conclusively, combined application of the fungal formulation with pyridaben at the low rate is a potential alternative for sustainable control of the mite pest in orchards of southern China.Field efficacies of B. bassiana and M. anisoplae formulations against cotton spider mites. Emulsifiable formulations of five isolates of B. bassiana and M. anisoplae were separately applied alone (Trial 1) or in conjunction with low rate of chlorpyrfos 48% EC at 10% of its labeled application rate (Trial 2) for control of spider mites (Tetranychus spp.) in cotton fields of Alaer, Xinjiang, northwest China during summer 2006. In Trial 1, randomized cotton plots (6×8 m) were sprayed twice with each of the five formulations alone at 15-day interval. In Trial 2, the plots were sprayed at the same rate of each formulation alone or together with chlorpyrfos at the low rate of 7.2 AI g ha^-1. Significant, but different, controls of spider mites by the fungal formulations were achieved after the first spray in Trial 1 and enhanced by the second. Ma456 alone resulted in an overall mean efficacy of 86% (63-96%) during Trial 1 and 88% (83-94%) during Trial 2. The best control occurred in the plots of Ma456 sprayed together with the low chemical rate, where mite densities on days 5 and 10 after spray declined by 92.4 and 93.9%, yielding the relative efficacies of 95.4 and 96.4% respectively. The formulation of Bb734 also provided desirable control in both trials. In Trial 2, mite density decline and relative efficacy were estimated as 81.0 and 89.1% on day 5 after the combined spray of Bb734 with chlorpyrfos and 83.9 and 91.1% on day 10, respectively. Microclimates in cotton canopy were featured with daily mean temperatures of 18.1-26.9℃(13.5-38.7℃) and the records of≥95% RH for 349 h during Trial 1 (35 days) and of 20.1-26.6℃(13.4-37.1℃) and≥95% RH for 298 h during Trial 2 (30 days). Thus, both formulations were highly efficacious for the spider mite control in cotton fields when applied alone and more efficacious when applied together with the low rate of chlorpyrfos.In summary, two bioassay systems were developed for evaluating efficiently the virulence of fungal biocontrol agents against eggs and adults of spider mites, and applied to successful selection of biocontrol candidates from different fungal species and isolates. Some acaricides biologically compatible with B. bassiana were also found for detecting the lethal effect of fungal/chemical interactions on spider mites in laboratory and for combined application of both agents for enhanced efficacy in the field. Emulsifiable formulations of at least some of the selected fungal candidates have proven promising for effective control of spider mites in citrus orchards of southern China or in cotton fields of northwest China. All the results would facilitate incorporation of fungal formulations into mite pest management systems at greatly reduced application rates of chemical acaricides.