Ecology of Metarhizium Anisopliae in Soilless Potting Media and the Rhizosphere: Implications for Pest Management

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Biological Control 32 (2005) 155–163 www.elsevier.com/locate/ybcon

Ecology of Metarhizium anisopliae in soilless potting media and the rhizosphere: implications for pest management

Denny J. Bruck¤

USDA-ARS, Horticultural Crops Research Laboratory, 3420 NW Orchard Avenue, Corvallis, OR 97330, United States

Received 8 July 2004; accepted 14 September 2004

Abstract

Wholesale container-grown ornamentals are often maintained at the nursery for at least two growing seasons and are subject to infestation by black vine weevil (BVW), Otiorhynchus sulcatus F., for several months each year. Therefore, a potting media amendment aimed at controlling BVW needs to persist for an extended period of time. These studies were conducted to determine the persistence and ecology of Metarhizium anisopliae (MetchnikoV) Sorokin incorporated into peat and bark-based potting media with and without a crab meal amendment in container-grown Picea abies ‘Nidiformis.’ Rooted cuttings of P. abies were planted into potting media amended with M. anisopliae (1 g of formulated product/L; »6 log10 CFU/g dry potting media). The fungal population in bulk potting media was quantitatively determined using selective media at 14, 21, 28, 35, 49, 63, 77, 91, 105, 119, 143, 175, 203, 231, 258, 287 and 342 days. The fungal population in the rhizosphere was quantitatively determined at 203, 231, 258, 287, and 342 days. M. anisopliae colonized the rhizosphere of P. abies and the fungal population in the rhizosphere was signiWcantly greater than in the surrounding bulk media. M. anisopliae persisted in the peat and bark-based potting media at 6.22 and 5.74 log10 CFU/g dry potting media for 342 days, respectively. Bioassays using bark and peat-based potting media inoculated with M. anisopliae at 6 log10 CFU/g dry potting media resulted in 93.5% and 97.5% infection of last instar BVW, respectively. P. abies roots inoculated with M. anisopliae infected 76% of 2nd–3rd instar BVW. Inoculation of roots with M. anisopliae represents a novel method for delivering entomopathogenic fungi and would greatly reduce application costs. Factors associated with fungal biology outside the host may be more important than virulence in a laboratory bioassay.  2004 Elsevier Inc. All rights reserved.

Keywords: Metarhizium anisopliae; Otiorhynchus sulcatus; Fungal biology; Rhizosphere colonization

1. Introduction the environment in the absence of their host in hopes that they will persist and infect their target once the host The inconsistent performance of biological control immigrates into the treated area. One system utilizing agents is often associated with an incomplete under- such an approach is the use of Metarhizium anisopliae standing of the ecological constraints of the biological (MetchnikoV) Sorokin (Hypocreales: Clavicipitaceae) to system in which they are placed. This is particularly true control the black vine weevil (BVW), Otiorhynchus sulc- for entomopathogenic fungi. There is little or no knowl- atus F., (Coleoptera: Curculionidae). The BVW is a edge of their biology outside of their insect host. How- polyphagous insect that is a severe pest of Weld-and con- ever, these fungi are often inundatively introduced into tainer-grown ornamentals as well as small fruit crops worldwide (Moorhouse et al., 1992). Economic losses are mostly associated with poor plant growth due to larval * Fax: +1 541 738 4025. root feeding and lost shipments due to quarantine issues. E-mail address: [email protected]. Other losses are associated with the cost of control, the

1049-9644/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2004.09.003 156 D.J. Bruck / Biological Control 32 (2005) 155–163 destructive sampling needed for larval scouting and cos- proliferate in the environment they are introduced (Nel- metic quality reduction due to leaf feeding (adult notch- son et al., 1994). Proliferation of rhizosphere competent ing of leaves). microbes for the biological control of soil borne plant Metarhizium anisopliae has been studied extensively diseases has been demonstrated using fungi (Harman, for the biological control of a wide range of insect pests, 1992) and bacteria (Kloepper and Beauchamp, 1992). including BVW (Booth and Shanks, 1998; Moorhouse For consistent use of microbes for biological control of et al., 1993a,b; Poprawski et al., 1985; Soares et al., 1983) plant diseases, it is essential that the biology and ecology and various other soil borne pests such as Popillia japon- of the biological control agent be understood completely ica (Coleoptera: Scarabaeidae) (Villani et al., 1994), (Lo et al., 1998). An understanding of the parameters Ligyrus subtropicus (Coleoptera: Scarabaeidae) (Raid impacting the ability of M. anisopliae to proliferate, and Cherry, 1992), Antitrogus parvulus (Coleoptera: maintain an eYcacious population and persist in the Scarabaeidae) (Samuels et al., 1990), Adoryphorus cou- microenvironment where control is desired would loni (Coleoptera: Scarabaeidae) (Rath, 1988) and Diab- improve biological control eYcacy. rotica undecimpunctata (Coleoptera: Chrysomelidae) All Wlamentous fungi with chitin as a major cell wall (Krueger and Roberts, 1997). The use, however, of M. component produce chitinases at all periods of active anisopliae and other entomopathogenic fungi for control growth (Gooday et al., 1992). Several insect pathogens of soil borne insects is often inconsistent due to limited including M. anisopliae, M. Xavoviride Gams and Rozsy- spore redistribution and persistence (Storey and Gard- opal (Hypocreales: Clavicipitaceae) and B. bassiana pro- ner, 1987, 1988; Storey et al., 1989). duce a complex mixture of chitinolytic enzymes during Increased understanding of the ecology of entomo- growth on insect cuticle (St. Leger et al., 1996). The pro- pathogens outside their host has enhanced our ability to duction of chitinase by M. anisopliae is dependent on eVectively utilize entomopathogens as biological control chitin availability (de Siqueria Pinto et al., 1997; St. agents. For example, Lewis and colleagues (Bing and Leger et al., 1996). M. anisopliae produces a heteroge- Lewis, 1991, 1992) observed that Beauveria bassiana (Bal- nous array of chitinases which may play a role in its abil- samo) Vuillemin (Hypocreales: Clavicipitaceae) grows ity to adapt to diVerent environments (St. Leger et al., endophytically within the green tissues of Zea mays L. 1993). (Cyperales: Poaceae). They then demonstrated that endo- The objectives of these studies were to quantify the phyte forming isolates of B. bassiana eVectively managed population of M. anisopliae over time in peat and bark- populations of the European corn borer, Ostrinia nubil- based potting media, determine the eVect of crab meal alis (Lepidoptera: Crambidae) (Lewis et al., 2002) while amendment (source of chitin) on the population size and being non-pathogenic to Z. mays (Lewis et al., 2001). persistence of M. anisopliae, determine if M. anisopliae Isolates of entomopathogenic fungi traditionally have colonized the rhizosphere of Picea abies (L.) Karst. been selected for development as biological control (Pinales: Pinaceae) ‘Nidiformis’ and determine if roots agents based on bioassay results from the laboratory inoculated with M. anisopliae were able to protect plants with little emphasis on understanding other fungal traits. from BVW larval feeding. Recently, an isolate of M. anisopliae (ARSEF 1080) was demonstrated to be rhizosphere competent (Hu and St. Leger, 2002). While the notion of fungal populations 2. Materials and methods increasing in the rhizosphere is not new, this was the Wrst report of an entomopathogenic fungus doing so. In Weld Two types of soilless potting media typically used in studies, the population of M. anisopliae in bulk soil container grown nursery production were used in the decreased from 105 to 103 propagules/g soil after several experiments. A 2:1 mixture of peat moss (Sunshine Mix months, while populations in the inner rhizosphere of #3, Sun Gro Horticulture, Bellevue, WA) and turkey grit cabbage plants remained at 105 propagules/g soil (Hu (Cherry Stone Grit #3, New Ulm, MN) and a bark-based and St. Leger, 2002). A signiWcantly larger fungal popu- media (OBC Northwest Nursery Mix #1, OBC North- lation in the inner rhizosphere compared to the outer west, Canby, OR) which consists of 70% Wne bark, 20% rhizosphere suggests that response to root exudates is mulch, 10% pumice. A commercial formulation of M. ani- involved in the rhizosphere eVect (Hu and St. Leger, sopliae (strain F52) [Earth BioSciences (New Haven, CT 2002) or that sporulation is enhanced in the rhizosphere. [formally Taensa Company])] was used. The formulated Root-deposited photosynthate is an important source of product consisted of M. anisopliae that had sporulated on readily available carbon for microbes in the rhizosphere rice grains and was then dried. The crab meal amendment (Butler et al., 2003). Rhizosphere competence is an attri- used was Eco-Logic (AgriGulf, Bayou La Batre, AL). bute of a microbe but the rate of colonization also The experiment was arranged as a randomized com- depends on the traits of the host (O’Connell et al., 1996). plete block in a split–split plot design with four replica- Biological control agents diVer fundamentally from tions. The whole plot was the type of soilless potting chemical agents in that in order to be eVective, they must media (peat, bark), the sub-plot the M. anisopliae D.J. Bruck / Biological Control 32 (2005) 155–163 157 application (control, incorporated granules, premix) using a spiral plater (iUL Instruments, Barcelona, Spain) incorporated at the rate of 1 g of formulated product/L of onto two plates of media selective for M. anisopliae (Veen potting media and the sub–sub plot the rate of crab meal and Ferron, 1966). Plates were incubated in complete amendment (0, 0.68 or 1.36 kg/yd3 of potting media). darkness at 28 °C for 4 days. The number of CFU/g dry bulk media was averaged across replicate plates for each 2.1. Media preparation sample. To ensure that the colonies counted were M. anisopliae, a total of 20 colonies morphologically identical to All experimental treatments were prepared by mixing those counted as M. anisopliae were randomly selected the appropriate ingredients in a 4 L twin-shell blender from a number of diVerent plates on each sample date (Patterson-Kelley, East Stroudsburg, PA). Each treat- and aseptically transferred to potato dextrose agar ment was mixed for 7 min to ensure that the fungus and/ (PDA) and allowed to sporulate. The colonies transferred or crab meal amendment were incorporated uniformly. were identiWed based on macro and microscopic charac- Seven days prior to planting, the premix treatment teristics (Humber, 1997). All colonies transferred to PDA was prepared by incorporating the full allotment of M. throughout the study were M. anisopliae. anisopliae and crab meal into one-third the total volume of potting media required for each treatment. These con- 2.3. Rhizosphere processing centrated mixtures (premix) were allowed to incubate in plastic pails covered with aluminum foil in the green- Based upon the report of M. anisopliae as a rhizo- house head house for 7 days. Ambient temperatures in sphere competent organism (Hu and St. Leger, 2002), the the head house were approximately 24–28 °C. After ability of F52 to colonize the rhizosphere of P. abies was 7 days, the premix was mixed with the remaining potting assessed beginning with the sample collected on day 203. media for each respective treatment. The premix proce- In the context of these studies, I used the deWnition of dure was performed to provide M. anisopliae the chance rhizosphere competence proposed by Schmidt (1979) to germinate and begin to grow, thus potentially increas- who deWned “rhizosphere competent” microorganisms ing the overall inoculum level in the potting media. as those that show enhanced growth in response to Rooted cuttings of P. abies ‘Nidiformis’ were potted developing roots or a classical rhizosphere eVect. The with the media from each treatment into 8.9 £ 8.9 £ rhizosphere population (CFU/g dry rhizosphere media) 8.9 cm pots (McConkey, Wilsonville, OR) and main- was determined from treatments of both potting media tained in a screenhouse where they were exposed to types, M. anisopliae applications and amendments of 0 ambient conditions. Each treatment consisted of 17 and 0.68 kg crab meal/yd3. To quantify the fungal popu- plants, one of which was selected randomly and destruc- lation in the rhizosphere, harvested plants (see above) tively sampled on each sample date. were shaken gently until only media tightly adhering to the root remained (rhizosphere). Plants were cut at their 2.2. Bulk media processing bases and the above ground portion discarded. The roots were placed into a sterile plastic bag (Nasco, Bulk media were sampled at 14, 21, 28, 35, 49, 63, 77, Modesto, CA) and held at 4 °C until processed (2– 91, 105, 119, 143, 175, 203, 231, 258, 287, and 342 days 3 days). M. anisopliae does not germinate or grow at after potting. The monthly maximum, minimum and temperatures at or below 4 °C (Ekesi et al., 1999; Halls- mean temperatures, respectively, for samples collected worth and Magan, 1999). To quantify the M. anisopliae days 14, 21, 28, and 35 (28.3, 9.7, and 19.1 °C), days 49 population in the rhizosphere, the entire root system and 63 (26.6, 8.3, and 17.2 °C), days 77 and 91 (18.7, 4.1, from each plant was placed into a plastic 250 ml Erlem- and 11.4 °C), days 105 and 119 (12.9, 2.3, and 7.6 °C), day yer Xask containing 90 ml of 0.05% Tween 80 solution 143 (9.7, 2.8, and 6.2 °C), day 175 (10.9, 2.8, and 6.9 °C), and processed as above. To quantify the amount of rhi- day 203 (11.5, 1.5, and 6.5 °C), day 231 (13.6, 5.2, and zosphere media on the root system of each plant sam- 9.4 °C), day 258 (14.4, 4.9, and 9.6 °C), day 287 (19.5, 6.5, pled, the suspension remaining in each Xask (once the and 13.0 °C), and 342 (25.5, 9.5, and 17.5 °C). Due to the roots were removed) was poured into a pre-weighed alu- large number of samples, two replications were sampled minum pan. Each Xask was carefully Xushed with dis- and processed completely on subsequent days. Plants tilled water to remove all soil particles. Pans containing were removed from the potting media by grasping the top the suspension were placed in a 38 °C drying oven until of the plant and gently pulling as the contents of the pot dry (approximately 24 h) and weighed. were dumped out and set aside. Ten grams of bulk potting media were placed in a plastic 250 ml Erlemyer Xask 2.4. Larval bioassay containing 90 ml of 0.05% Tween 80 solution, shaken (250 rpm) for 20 min at room temperature, then placed in A laboratory bioassay was performed at the conclu- an ultrasonic cleaner (Model 5210, Branson Ultrasonic, sion of the above described experiment with the peat and Danbury, CT) for 2 min. Serial dilutions were plated bark-based media without a crab meal amendment. 158 D.J. Bruck / Biological Control 32 (2005) 155–163

Potting media of each type were prepared (as described 2.6. Data analysis previously) with a target concentration of 1 £ 106 spores of M. anisopliae (strain F52)/g dry potting media. The Analysis of data from the bulk media sampling was purpose of the bioassay was to determine if the mean performed using the General Linear Models Procedure fungal inoculum observed in both potting media for the (GLM) with Tukey’s multiple range test used to separate duration of the above experiment was adequate to infect means (SAS Institute, 1999). The data from each of the last instar BVW. The peat and bark-based media were 17 sampling dates were analyzed separately. M. anisop- inoculated with grains of rice on which the M. anisopliae liae was never isolated from the control treatment and had been allowed to sporulate. Each treatment was these data were removed when calculating the mean fun- mixed in 4 L twin-shell blenders as described previously. gal population (log10 CFU/g dry potting media). The To verify the inoculation rate, samples of each treatment data were analyzed as a split–split plot design with were immediately taken and the CFU/g dry potting hypothesis testing used to pair the correct error terms in media determined as described previously. The mean the F test. Although samples were collected from each spore concentration in the bioassay was 1.87 £ 106 CFU/ treatment over time, a repeated measures analysis was g dry potting media (determined via spiral plater). not required because plants were maintained individu- Approximately 200 cm3 of the inoculated potting media ally, destructively sampled and the fungal population was placed in 236 cm3 plastic containers along with 20 from each pot quantiWed only once. last-instar BVW and a carrot slice as a food source. The A one sample t test was performed to determine if the V cups were incubated in complete darkness at 24 °C and di erence between the log10 CFU/g dry potting media in larval mortality recorded at 14 days. Larvae were the rhizosphere and bulk media of each plant sampled obtained from a laboratory colony maintained at the was signiWcantly diVerent from zero (SAS Institute, USDA-ARS, Horticultural Crops Research Laboratory, 1999). The t test revealed that on each sample date, the V Corvallis, OR. The experiment was arranged in a ran- di erence between the mean log10 CFU/g dry soil in the domized complete block design, replicated four times rhizosphere and bulk media was signiWcantly greater and included an untreated control for both potting than zero. These diVerence data were then analyzed media types. The experiment was performed twice. using GLM and Tukey’s multiple range test used to separate means (SAS Institute, 1999). 2.5. Root inoculation Data from the larval bioassays and root inoculation studies were analyzed using GLM and t test used to sep- Rooted cuttings of P. abies ‘Nidiformis’ were dipped arate means (SAS Institute, 1999). A test of homogeneity into 400 ml of a 0.05% Tween 80 solution containing of variance was performed to detect variation between 2 £ 106 spores/ml of M. anisopliae (strain F52), placed on the two runs of each experiment (Little and Hills, 1978). sterile paper towel on the laboratory bench top and Variability was not signiWcant between runs of either allowed to dry. The roots of the cuttings were potted experiment and the data from the runs were combined with the 2:1 mixture of peat moss and turkey grit for analysis. (described above) and placed in petri dishes (100 £ 30 mm) with a small opening for the plant stem. The petri dishes served as small rhizotrons which 3. Results allowed for the observation of larvae feeding on the roots as well as any larvae that became infected with M. 3.1. Fungal persistence anisopliae. The plants were maintained in an upright position in the greenhouse (27 § 3°C) for 5days and Regression analysis was used to describe the relation- W watered as needed. After 5 days, ve 2nd–3rd instar ship between the M. anisopliae population (log10 CFU/g BVW larvae from the laboratory colony were added to dry potting media) and sample date in both potting each dish and the plants returned to the greenhouse. The media. Throughout the course of the study there was a experiment was performed in a completely randomized signiWcant decline in the fungal population in both the 2 design with six replications. Control plants were dipped peat-based (log10 CFU D 6.69 ¡ 0.0016 d; R D 0.83) and into 400 ml of 0.05% Tween 80 solution only and BVW bark-based potting media (log10 CFU D 6.63 ¡ 0.0036 d; larvae added in the same manner. The experiment was R2 D 0.80). The decline in fungal density in both media performed twice with individual batches of spore and types was very gradual, particularly in the peat-based control suspensions. Thirteen days after larvae were potting media. There were no signiWcant diVerences in placed in the petri dishes, larvae were observed through fungal density between media types for the Wrst 119 days the lid and base of the petri dish on treated plant roots post-treatment (Fig. 1). The fungal density in both types infected with M. anisopliae. At this point, the plants were of potting media nearly mirrored one another the Wrst returned to the laboratory and the contents of each dish 119 days post-application with no signiWcant diVerence carefully searched for infected or healthy BVW larvae. in fungal population between the two potting media. D.J. Bruck / Biological Control 32 (2005) 155–163 159

Fig. 1. Mean log10 CFU/g of dry potting media (§SD) (with the control treatments removed from the analysis) of Metarhizium anisopliae (strain F52) in peat and bark-based potting media for 342 days. Points denoted with an (*) are signiWcantly diVerent (P < 0.05).

Following the marked decline in fungal density in the types of fungal application were very gradual. While the bark-based media on day 143, the fungal density in the rate of decline in fungal population was very similar in peat-based media was signiWcantly higher on each of the the incorporated and premix applications, the fungal subsequent sample dates (Fig. 1). population in the premix application was signiWcantly While the fungal inoculum load in the potting media higher initially and this diVerence was maintained over was not immediately quantiWed after fungal incorpora- the 342 days of the study. tion at the beginning of the persistence study, there appears to have been an initial increase in the fungal 3.2. Larval control population in the premix treatment (as hypothesized) that was sustained throughout the course of the experi- Incorporation of 1.87 £ 106 CFU/g dry potting media W ment. The fungal population (log10 CFU/g dry potting resulted in signi cant levels of larval infection media) appears to have increased signiWcantly during the (P < 0.001). The levels of last instar BVW infection with 7-day incubation period as indicated by the signiWcantly M. anisopliae in the bark and peat-based potting media higher fungal population in the premix treatment on day were 93.5 and 97.5%, respectively. 14 (Fig. 2). When the aluminum foil covers were removed from the top of the pails containing the concen- 3.3. Rhizosphere competence trated fungal mixture, M. anisopliae hyphal growth was visibly evident in the potting media. The fungal popula- The one sample t test revealed that on each sample tion in both treatments followed a similar pattern, how- date the diVerence between the M. anisopliae population ever, the population in the premix treatment was in the rhizosphere and surrounding bulk media was sig- W W signi cantly higher (approximately 0.5 log10 CFU/g dry ni cantly greater than zero (Table 1). These data indi- potting media) throughout the course of the experiment cate that not only did M. anisopliae colonize the (Fig. 2). Regression analysis was used to describe the rhizosphere of P. abies, but the M. anisopliae population relationship between the M. anisopliae population (log10 responded favorably to the rhizosphere microclimate. CFU/g dry potting media) and sample date for both The mean diVerence in M. anisopliae population in the W means of fungal application. There were a signi cant rhizosphere ranged from 0.65 to 1.28 log10 CFU/g media. declines in the fungal population in both the incorpo- The GLM analysis of the data consisting of the mean 2 V rated (log10 CFU D 6.43 ¡ 0.0029 d; R D 0.87) and di erence between the rhizosphere and bulk media pop- 2 premix (log10 CFU D 6.90 ¡ 0.0023 d; R D 0.77) applica- ulation from each plant sampled showed that potting tions. The declines in fungal population utilizing both media type was the only parameter studied that had any 160 D.J. Bruck / Biological Control 32 (2005) 155–163

Fig. 2. Mean log10 CFU/g of dry potting media (§SD) of Metarhizium anisopliae (strain F52) averaged over peat and bark-based potting media in each of the fungal incorporation treatments (with the control treatments removed from the analysis). Incorporated—1 g of formulated fungal granules/L of potting media; Premix—Wnal concentration of 1 g of formulated fungal granules/L of potting media. A premix (a third of the total volume of potting media for each treatment inoculated with the total amount of fungal granules) were allowed to incubate in plastic pails in the greenhouse head house for 7 days covered with aluminum foil. This premix was added to the remaining volume of potting media for each respective treatment. Points denoted with an (*) are signiWcantly diVerent (P < 0.05).

Table 1 3.4. Root inoculation Mean diVerence (§SD) between the rhizosphere and bulk media Meta- rhizium anisopliae populations (log10 CFU/g dry potting media) and the Inoculation of roots of P. abies with 2 £ 106 spores/ml probability that the observed diVerence was greater than zero of M. anisopliae was a successful fungal delivery system. a V b Day Mean di erence t statistic df P > t A signiWcant percentage (§SEM) (76 § 9%) of the BVW 203 0.65 § 0.48 10.73 30 c <0.001 larvae feeding on the fungal-treated roots were infected 231 0.71 § 0.64 8.76 31 <0.001 with M. anisopliae (P < 0.001). There were no larvae 258 0.83 § 0.32 19.47 31 <0.001 287 1.28 § 0.48 22.26 31 <0.001 infected with M. anisopliae in the control treatment. All 342 0.97 § 0.32 23.48 31 <0.001 of the infected larvae were located near the root. The a Number of days post-fungal application that samples were taken. roots of the plants where infected larvae were found b A one sample t test performed to determine if the diVerence were visually inspected for signs of larval feeding. Initial between the log10 CFU/g dry potting media in the rhizosphere and bulk larval feeding was often observed where larvae had media of each plant sampled was signiWcantly diVerent than zero (SAS located plant roots and begun to feed before succumbing Institute, 1999). to infection. These data demonstrate that inoculation of c One less degree of freedom than expected due to contaminated plates from one sample. plant roots with a rhizosphere competent isolate of M. anisopliae is a useful, feasible approach for managing BVW and perhaps other root-feeding insects. signiWcant eVect on the size of the diVerence observed. The diVerence in M. anisopliae population between the rhizosphere and bulk media was greatest in the peat- 4. Discussion based potting media on three of the Wve sample dates (Table 2). There were no signiWcant diVerences in the The ability of M. anisopliae-treated roots to control mean diVerence between the rhizosphere and bulk media BVW larvae indicates the potential to use colonized roots M. anisopliae populations due to fungal application or as a delivery system for fungal biological control agents crab meal amendment, nor were there any signiWcant of root-feeding insects. There is a large volume of pub- interactions. lished work in the Weld of plant pathology on the use of D.J. Bruck / Biological Control 32 (2005) 155–163 161

Table 2 development and thus come into contact with the rhizo- Mean diVerence and 95% conWdence interval between the rhizosphere sphere competent fungus and become infected. By using and bulk media Metarhizium anisopliae populations (log10 CFU/g dry this approach, the application of entomopathogenic fungi potting media) due to potting media type should be economical as only the roots of small plants a c d Day Potting Mean Lower Upper (rooted cuttings, tissue culture) or potentially seeds would media diVerence b have to be inoculated. If rhizosphere competent isolates of 203 Bark 0.44a 0.09 0.78 entomopathogenic fungi actively colonize elongating Peat 0.82a 0.67 0.97 roots and persist with the growing plant, plants could be 231 Bark 0.38a 0.17 0.58 protected for extended periods of time. Peat 1.04b 0.83 1.24 In these studies were the M. anisopliae rhizosphere 258 Bark 0.69a 0.55 0.83 population was quantiWed, plants were grown in potting Peat 0.97b 0.83 1.11 media in which M. anisopliae was uniformly incorpo- 287 Bark 1.14a 0.90 1.38 rated. Therefore, the entire root system was in contact Peat 1.43a 1.19 1.67 with an inoculum source throughout the length of the 342 Bark 0.87a 0.76 0.98 study. The ability of M. anisopliae to colonize elongating Peat 1.08b 0.97 1.19 roots is unclear. M. anisopliae was isolated at a soil a Number of days post-fungal application that samples were taken. depth of 10 cm from plants in which the inoculum was b V GLM used to determine if the di erence between the log10 CFU/g applied as spore suspension on the soil surface, suggest- dry potting media in the rhizosphere and bulk media of each plant ing some degree of vertical movement either through sampled was signiWcantly diVerent on each individual sample date. Means separated with Tukey’s multiple range test (SAS Institute, colonization or via percolating water (Hu and St. Leger, 1999). 2002). The largest population of M. anisopliae in the c Lower limit of the 95% conWdence interval of the mean (SAS inner and outer rhizosphere was observed in the Wrst Institute, 1999). 2 cm of root and declined signiWcantly from 2 to 10 cm d W Upper limit of the 95% con dence interval of the mean (SAS (Hu and St. Leger, 2002). Most of the M. anisopliae inoc- Institute, 1999). ulom on cabbage roots was easily removed by washing which suggests weak adhesion to the root (Hu and St. Leger, 2002) or that spores are easily removed. rhizosphere competent organisms (both bacteria and Therefore, the criteria for deWning a rhizosphere com- fungi) for the microbial control of plant diseases (Har- petent entomopathogenic fungus will have to evolve to man, 1992; Kloepper and Beauchamp, 1992). This pro- keep pace with the goals and methodology in mind when vides a wealth of knowledge which can be used to develop developing them. The key concept of root colonization the use of rhizosphere competent entomopathogenic by beneWcial bacteria is that the bacteria grow in the fungi for pest control. When selecting an isolate, under- presence of natural Xora (Schroth and Hancock, 1982) standing factors associated with entomopathogen biology and therefore are competitive with bacteria and fungi outside their insect host may be more important than its present in the soil. More recently the deWnition of “rhi- virulence in a laboratory bioassay of insect infection. zosphere competence” has been reWned when consider- Currently, when protecting plants from root-feeding ing biological control agents to, “the ability of a insects using entomopathogenic fungi, eVorts are concen- microorganism, applied by seed treatment, to colonize trated on applying large amounts of inoculum to increase the rhizosphere of developing roots” (Baker, 1991). A the fungal population throughout the bulk soil. This tech- deWnition for rhizosphere competent entomopathogenic nique presents numerous problems including: (1) a fungi would require that the fungus grow and persist in requirement for large quantities of fungal inoculum, often the presence of natural Xora in the soil or potting media making applications uneconomical, (2) it is diYcult to get and have the ability to colonize the rhizosphere around the fungal propagules applied to the soil surface to pene- developing roots at populations great enough to infect trate into the soil more than a few centimeters and (3) a pest insects feeding on the root. large amount of time, money and eVort is spent protecting Metarhizium anisopliae was also shown to persist well areas of the bulk soil where the pest either does not occur in both peat and bark-based bulk potting media for up or is not of concern. By developing techniques to use the to 342 days. The ability of M. anisopliae to persist in hor- roots as a delivery system for entomopathogenic fungi, the ticultural potting media (often microbially impoverished costs and logistics of biological control would be much because of the acidic peat content) may not translate more favorable. As demonstrated here, it is possible to use well in microbially diverse Weld soils. The cause for the roots treated with levels of entomopathogenic fungal inoc- marked reduction in fungal density in the bark-based ulum comparable to those colonizing the rhizosphere to media on day 143 is unclear. Moisture retention might infect insects and protect the plant from root feeding. be one of the most signiWcant factors aVecting microbial Regardless of the root-feeding insect considered, they all activity (GriYn, 1969, 1981). Soil temperature also can must feed on plant roots in order to complete their impact the survival and infectivity of M. anisopliae in 162 D.J. Bruck / Biological Control 32 (2005) 155–163 soil (Ekesi et al., 2003; Li and Holdom, 1993). Fungal improved the manuscript. This work was supported persistence generally is reduced in soils with higher water solely by the United States Department of Agriculture, contents. The samples collected on days 119 and 143 cor- Agricultural Research Service, PaciWc West Area, Horti- respond to November 18th and December 12th, 2003, cultural Crops Research Laboratory, Corvallis, Oregon, respectively. Ambient winter conditions in Oregon are CRIS # 5358-22000-029-00D. Mention of trade names cool and damp which may have allowed the bark-based or commercial products in this publication is solely for potting media to remain wet for a prolonged period. the purpose of providing speciWc information and does Decline of M. anisopliae in wet soils (0 and ¡2.0 kPa) not imply recommendation or endorsement by the US occurs at 30 and 60 days in clay and sandy loam soil, Department of Agriculture. respectively (Li and Holdom, 1993). The results of the larval bioassays indicate that the References mean level of inoculum (»6log10 CFU/g dry potting media) observed in both potting media types over the Y Baker, R., 1991. Induction of rhizosphere competence in the biological duration of the experiment was su cient to provide control fungus Trichoderma. In: Keister, D.L., Cregan, P.B. (Eds.), excellent control of last instar BVW. This indicates that The Rhizosphere and Plant Growth. 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Potential of a dried rice/mycelium The use of a premix treatment would also provide formulation of entomopathogenic fungi to suppress subterranean W pests in small fruits. Biocontrol Sci. Technol. 8, 197–206. bene ts to growers wishing to employ M. anisopliae in Butler, J.L., Williams, M.A., Bottomley, P.J., Myrold, D.D., 2003. their BVW control program. This type of program Microbial community dynamics associated with rhizosphere car- would be particularly useful for plants with roots that bon Xow. Appl. Environ. Microbiol. 69, 6793–6800. are not colonized by M. anisopliae or for those plants de Siqueria Pinto, A., Barreto, C.C., Schrank, A.C., Ulhoa, J., Vain- which rhizosphere colonization had not been demon- stein, M.H., 1997. PuriWcation and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae. strated. The amount of fungal product required to Can. J. 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Water and temperature relations of cally have not been very successful, and when success growth of the entomogenous fungi Beauveria bassiana, Metarhiz- has been demonstrated, it has not been consistent. A ium anisopliae, and Paecilomyces farinosus. J. Invertebr. Pathol. 74, completely new approach which shifts the focus of our 261–266. eVorts away from laboratory bioassay data and onto Harman, G.E., 1992. Development and beneWts of rhizosphere compe- fungal ecology may very well lead to success. tent fungi for biological control of plant pathogens. J. Plant Nutr. 15, 835–843. Hu, G., St. Leger, R., 2002. Field studies using a recombinant mycoin- secticide (Metarhizium anisopliae) reveal that it is rhizosphere com- Acknowledgments petent. Appl. Environ. Microbiol. 68, 6383–6387. Humber, R.A., 1997. Fungi: identiWcation. In: Lacey, L.A. (Ed.), Man- I thank Kelly Donahue for all of her hard work in ual of Techniques in Insect Pathology. 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