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Biological Control 49 (2009) 610

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Biological Control
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The effects of Beauveria bassiana dose and exposure duration on colonization and growth of tissue cultured banana (Musa sp.) plants
Juliet Akello a,b, Thomas Dubois a,*, Daniel Coyne a, Samuel Kyamanywa b
a b

International Institute of Tropical Agriculture (IITA), Lambourn & Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK Faculty of Agriculture, Makerere University, P.O. Box 7062, Kampala, Uganda

a r t i c l e

i n f o

a b s t r a c t
Beauveria bassiana (Balsamo) Vuillemin, an entomopathogenic fungus, can exist asymptomatically as an endophyte in banana (Musa spp.) and potentially provide management against the banana weevil Cosmopolites sordidus (Germar). However, in order to optimize its biological control potential when deployed as an endophyte, it is essential to establish the inoculum dose and exposure duration that provides suitable levels and distribution of the pathogen within the plant. It is equally important to determine any negative tness costs that might be associated with increased population of the fungus in the host plant. In an attempt to optimize plant colonization by this fungus, the effects of B. bassiana doses and exposure durations on colonization and growth of tissue cultured banana (AAA-EA, cv. Kibuzi) plants were investigated. The plants were inoculated by dipping in B. bassiana suspension and then grown in the screenhouse for a month. B. bassiana successfully colonized banana plants with 96.7% of B. bassiana-treated plants colonized, 1 month after inoculation. The optimal dose and exposure duration for effective colonization of tissue cultured banana plants varied, but dipping plants in a dose of 1.5 107 conidia/ml for 2 h produced the best percentage colonization. Endophytic colonization of tissue cultured banana plants by B. bassiana had no negative impact on plant growth, even when plants were inoculated at the highest dose. 2009 Elsevier Inc. All rights reserved.

Article history: Received 10 January 2008 Accepted 6 June 2008 Available online 15 June 2008 Keywords: Banana plants Beauveria bassiana Biological control Conidial dose Cosmopolites sordidus Endophytic colonization

1. Introduction Bananas and plantains (Musa spp.) are among the most important subsistence food crops in the world. In East Africa, it is widely grown in all types of agricultural systems, from small, mixed, subsistence gardens, to large multinational commercial monocultures (INIBAP, 1986; Gold et al., 1993). However, bananas are affected by a complex of pests and diseases (Speijer and Fogain, 1999; Gold et al., 2004). The banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) remains among the most serious pest constraints to banana production in East Africa. Yield losses of up to 100% may result from banana weevil infestations (Koppenhofer et al., 1994; Gold et al., 2004). The control of C. sordidus has been based on use of cultural practices and chemical pesticides. However, cultural practices such as weevil trapping, hot water treatment, good crop husbandry and use of clean planting materials tend to be labor intensive and costly and have consequently shown only limited success (Gold et al., 2001, 2002; Masanza et al., 2005; De Graaf et al., 2007). Although chemical pesticides are quite effective, factors such as insect resistance, environmental pollution, cost and availability limit their use
* Corresponding author. Fax: +44 256 41285079. E-mail addresses: t.dubois@cgiar.og, sundayseed@yahoo.com (T. Dubois). 1049-9644/$ - see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2008.06.002

for management of C. sordidus (Collins et al., 1991; Gold et al., 1999). Biological control using natural enemies of C. sordidus has been investigated, but parasitism and predation levels were low and of no major importance to banana weevil population levels (Koppenhofer et al., 1992; Treverrow et al., 1993). The potential for use of the entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) against the banana weevil has been investigated by several researchers (Kaaya et al., 1993; Nankinga, 1994, 1999; Godonou et al., 2000). Despite being highly pathogenic to banana weevils in the laboratory, implementation in the eld has been less successful (Nankinga, 1999; Godonou et al., 2000; Schoeman and Schoeman, 1999; Schoeman and Botha, 2003). Abiotic factors, including rainfall, temperature, humidity, sunlight and pesticides and biotic factors, such as interactions with other microorganisms, adversely affect conidial viability (OCallaghan et al., 2001; Bruck and Lewis, 2002; Fuxa and Richter, 2004; Thompson et al., 2006), thus limiting its use in an integrated pest management scheme. In addition, current application methods of B. bassiana in banana elds remain impractical and expensive. At the International Institute of Tropical Agriculture (IITA), the potential of B. bassiana as an articial endophyte of banana plants is being investigated (Akello et al., 2007). Until recently, however, no documentation of B. bassiana colonization of banana plants had

J. Akello et al. / Biological Control 49 (2009) 610

been recorded. When tissue cultured banana plants (AAA-EA, cv. Kibuzi,) were dipped for 2 h in a B. bassiana (strain G41) suspension of 1.5 1010 conidia/ml, the fungus established as an endophyte in banana roots, rhizomes and pseudostem bases for at least 1 month after inoculation, without any negative tness costs on the host plant (Akello et al., 2007). In order to optimize its biological control potential against C. sordidus inside the plant, colonization levels might need to be relatively high, especially in the rhizome, which are mostly affected by the larvae, the most damaging stage of the banana weevil. On the other hand, negative plant effects may result from high colonization levels, thereby compromising plant growth. This study, therefore, aimed to determine optimal B. bassiana dose and exposure duration for effective plant colonization, which would not negatively affect plant growth. 2. Materials and methods 2.1. Experimental site and design A screenhouse experiment was conducted at IITA in Namulonge, Uganda, to determine the effects of varying dose and dipping duration on B. bassiana colonization of tissue cultured banana plants. The experiment was organized as a completely randomized design with a factorial arrangement. Factors included inoculum dose (1.5 105, 1.5 106, 1.5 107 and 1.5 108 conidia/ml) and exposure duration (1, 2 and 4 h). For each treatment, 10 plants were used and the experiment was conducted a total of three times. 2.2. Fungal inoculum Beauveria bassiana strain G41, obtained from the Kawanda Agricultural Research Institute of the National Agricultural Research Organization (NARO), Kawanda, Uganda was chosen for use throughout the study based on its pathogenicity against C. sordidus (Nankinga, 1999) and its ability to grow as an endophyte in banana (Akello et al., 2007). The fungus was originally isolated from soil from banana plantations and had been stored on silica gel at 21 24 C. The fungus was revived on Sabouraud dextrose agar medium supplemented with yeast extract (SDAY) (200 g glucose, 20 g peptone, 5 g yeast extract and 15 g agar/l distilled water) containing antibiotics (0.1 g/l penicillin, 0.2 g/l streptomycin and 0.05 g/l chlortetracycline SDAY) in 55 mm diameter plastic Petri dishes in the laboratory ($25 C and a natural photoperiod of $12:12 h L:D) for 21 days. From these cultures, B. bassiana suspension of 1.5 107 conidia/ml was prepared and ve female banana weevils immersed in the suspension for 10 min. The weevils were incubated inside a 90 mm diameter sterile Petri dish lined with a sterile, moistened lter paper for 2 weeks. Insect cadavers showing mycosis were removed from the dish, fungal mycelia scraped from the insect cadavers using a sterile scalpel blade and then dissolved in sterile 0.01% Tween 80. The resulting suspension was plated on SDAY supplemented with antibiotics. After 24 h, single spores were picked and transferred singly to ten 55 mm Petri dishes containing SDAY using a sterile needle. The Petri dishes were incubated in the laboratory for 21 days, after which they were stored at 4 C and used as a stock for generating inoculum. Conidial suspensions from single spore cultures, prepared in 0.01% Tween 80, were used to inoculate SDAY in 90 mm diameter plastic Petri dishes. Cultures were incubated in the laboratory for another 21 days. The Petri dish lids were removed under sterile conditions in a laminar air ow cabinet and the cultures air dried for 24 h prior to conidial harvest. During harvest, the cultures were scraped from the surface of the dried medium with a scalpel blade and sieved through a 150 lm sieve into sterile aluminum foil. The

harvested conidia were suspended in 500 ml sterile 0.01% Tween 80 in a sterile 180 70 mm bottle. For each treatment, conidial density was determined using an improved Neubauer haemocytometer and adjusted to 1.5 105, 1.5 106, 1.5 107 and 1.5 108 conidia/ml in 300 ml. 2.3. Plant inoculation Tissue cultured banana plants (cv. Kibuzi), generated axenically from shoot-tip cultures (Vuylsteke, 1998) were hardened in a humidity chamber for 4 weeks at 1932 C under non-axenic and natural light conditions. Each plant was transferred singly to a 250 ml nutrient solution containing 1 g/l Poly-Feed (Haifa chemicals, Haifa, Israel) in 300 ml plastic cups prior to placing in a humidity chamber. A sponge wrapped around the pseudostem base supported plants when placed in the nutrient solution through a hole in the lid. The nutrient solution was changed weekly. After 4 weeks, plants were removed from the nutrient solution and dipped in the four concentrations of conidia in a 300 ml suspension for 1, 2 or 4 h, corresponding to each of the treatments. Three control treatments comprised plants that were dipped in sterile 0.01% Tween 80 for 1, 2 or 4 h, respectively, while a fourth control treatment consisted of plants that were not dipped. Ten plants per treatment were used in the study. The plants were transferred singly to 3 L black polythene bags containing steamsterilized loamy forest soil. Plant height (the distance from the base of the plant to the youngest leaf axil), number of fully developed leaves, leaf width (widest part of the lamina) and leaf length (the distance from the leaf apex to the leaf stalk) of the youngest leaf were recorded immediately after inoculation. The plants were maintained in the screenhouse for 1 month and watered daily. 2.4. Harvest At 1 month after inoculation, plant height, number of fully developed leaves and width and length of the youngest leaf were recorded. Each plant was removed from the soil and washed using tap water to remove soil. Using a sterile blade, pseudostems were cut at $3 cm above the pseudostem base. Fresh shoot weight for each plant was recorded and, after drying the shoots in a hot air oven at 60 C for 48 h, dry shoot weight was recorded. Plant colonization was assessed through re-isolation of B. bassiana on modied SDAY (same medium indicated above but supplemented with 250 mg/l chloramphenicol, 0.75 mg/l 50% (w/w) benomyl (Benlate, Dupont, Wilmington, USA), 24 mg/l copper oxychloride) and 10 mg/l crystal violet) from three randomly selected root pieces ($10 cm in length), a rhizome piece and a pseudostem base piece per plant according to Akello et al. (2007). The roots, rhizome and pseudostem base pieces were surface sterilized under sterile conditions by dipping in 5% (active chlorine) NaOCl containing 0.05% Tween 20 for 1 min, followed by 75% EtOH for 1 min and rinsed thrice in sterile deionized water. Six segments from each root piece (0.40.5 cm long), rhizome piece and pseudostem base piece (0.20.4 cm3) were plated onto modied SDAY in one 55 mm diameter plastic Petri dish. Petri dishes were incubated for 2 weeks in the laboratory, after which all plant pieces were examined visually for fungal growth. Fungal growth was characterized as B. bassiana based on white dense mycelia, becoming cream to pale yellow at the edge (Humber, 1997). In situations where there was contamination or potential confusion with other fungal taxa, both mycelium and conidia were removed using a sterile needle and mounted in a drop of water on a microscope slide. The mounted slide was examined under the microscope for characteristic B. bassiana features (globose conidia and zigzag-shaped conidiophores) (Humber, 1997). For each root, rhizome and pseudostem

J. Akello et al. / Biological Control 49 (2009) 610

base piece, percentage colonization was calculated as number of segments exhibiting B. bassiana outgrowth per total number of segments. 2.5. Data analysis To obtain a normally distributed datum with homogeneous variance among treatments, plant height and leaf width at plant inoculation were square-root and log transformed, respectively. At harvest, plant height, leaf length and width, and fresh shoot weight were square-root transformed, while dry shoot weight was log transformed. All plant growth parameters were analyzed using analysis of variance (ANOVA), except for number of standing leaves that was analyzed using KruskalWallis tests. For parameters that were analyzed using ANOVA, treatment means were separated using Tukeys studentized range test, while for the number of standing leaves a Bonferroni test was used to separate the treatment means. In all experiments, percentage colonization of plant tissues was analyzed using logistic regression. Differences in percentage colonization among treatment means were separated using a Dunn Sidak correction (SAS Institute, 1995; Sokal and Rohlf, 1995). 3. Results 3.1. Plant colonization Beauveria bassiana successfully colonized tissue cultured banana plants, with 96.7% of B. bassiana-treated plants colonized by the fungus. None of the control plants were colonized by the fungus. Irrespective of plant part, a total of 10,800 segments were plated from B. bassiana inoculated plants and out of these 4496 segments were colonized by the fungus. Whereas 1176 segments were contaminated by fungi from other taxa and were treated as missing data, 5128 segments were not colonized at all by any fungus. Overall, percentage colonization was inuenced by inoculum dose (v2 = 72.91, df = 3, P < 0.0001). Increasing dose increased percentage colonization up to a dose of 1.5 107 conidia/ml and decreased when this dose was exceeded (Fig. 1). Percentage colonization was also inuenced by dipping duration (v2 = 7.92, df = 2, P = 0.019), with colonization rates of plants that were dipped for 2 (47.6%) and 4 (48.1%) h greater than for plants dipped for 1 h (44.4%) (P < 0.017). However, there was an interaction between dipping duration and inoculum dose (v2 = 30.77, df = 6, P < 0.0001). At low conidial dose of 1.5 105 conidia/ml, maximum colonization was attained when plants were dipped for a

longer period of time (4 h). At moderate doses of 1.5 106 and 1.5 107 conidia/ml, percentage colonization was not affected by dipping duration. At a higher dose of 1.5 108 conidia/ml, dipping plants for 1 and 2 h attained better percentage colonization than dipping for 4 h (Fig. 2). Percentage colonization varied signicantly among plant parts (v2 = 306.84, df = 2, P < 0.0001), with colonization of roots (52.6%) higher than rhizome (47.4%) and pseudostem base (30.7%) colonization (P 6 0.017). However, plant part colonization depended on inoculum dose (v2 = 72.05, df = 3, P < 0.0001) and dipping duration (v2 = 30.89, df = 6, P < 0.0001) (Tables 1 and 2). Whereas a dose of 1.5 107 conidia/ml provided the best percentage rhizome colonization, doses of 1.5 106 and 1.5 107 conidia/ml attained similar percentage pseudostem base and root colonization (P 6 0.0085). Although there was no effect of dipping duration on rhizome and pseudostem base colonization (v2 = 2.24, df = 3, P P 0.33), percentage colonization of plant roots was affected by dipping duration (v2 = 11.51, df = 2, P 6 0.0032). However, there was an interaction between dose and dipping duration on colonization of all plant parts (v2 = 34.00, df = 6, P < 0.0001) (Fig. 3). Irrespective of plant part, plants dipped for few hours (1 h) required a higher inoculum dose, while those dipped for longer hours (4 h) required lower inoculum doses. Hence, better root, rhizome and pseudostem base colonization was attained when plants were treated with doses of 1.5 108 conidia/ml and 1.5 105 conidia/ml for 1 and 4 h, respectively. Whereas exposure duration of 2 h had little effect on root and pseudostem base colonization, optimal percentage rhizome colonization was attained when plants were treated at a dose of 1.5 107 conidia/ml at this exposure duration.

100 90 80 70 60 50 40 30 20 10 0

Colonization (%)

1.5 10

1.5 10

1.5 10
1h

1.5 10
4h

Inoculum dose (conidia/ml)


2h

Colonization (%)

100 90 80 70 60 50 40 30 20 10 0

Fig. 2. The effect of dipping duration and inoculum dose on percentage colonization of tissue-cultured banana (cv. Kibuzi, Musa sp., genome group AAA-EA) plants, 1 month after inoculating with Beauveria bassiana strain G41. Colonization (%) represents the number of colonized segments divided by total number of cultured segments100.

a b

Table 1 Effect of inoculum dose on percentage colonization of tissue-cultured banana (cv. Kibuzi, Musa sp., genome group AAA-EA) rhizome, pseudostem base and root, 1 month after inoculating with Beauveria bassiana strain G41 Concentration (conidia/ml) % Colonization Rhizome Pseudostem base b b a b c 24.5 33.3 35.8 29.1 0.0 b a a b c Root 50.3 54.1 56.7 49.7 0.0 b ab a b c

1.5 10

1.5 10

1.5 10

1.5 10

Inoculum dose (conidia/ml)


Fig. 1. The effect of inoculum dose on percentage colonization of tissue-cultured banana (cv. Kibuzi, Musa sp., genome group AAA-EA) plants, 1 month after inoculating with Beauveria bassiana strain G41. Colonization (%) represents the number of colonized segments divided by total number of cultured segments100.

1.5 10 1.5 106 1.5 107 1.5 108 0

41.4 47.2 59.4 41.6 0.0

For each plant part, gures followed by similar letters are not signicantly different at P 6 0.0085 (Dunn Sidak correction factor).

J. Akello et al. / Biological Control 49 (2009) 610 Table 2 Effect of dipping duration on percentage colonization of tissue-cultured banana (cv. Kibuzi, Musa sp., genome group AAA-EA) rhizome, pseudostem base and root, 1 month after inoculating with Beauveria bassiana strain G41 Dipping duration (h) %Colonization Rhizome 0 1 2 4 0.0 45.8 49.6 47.0 b a a a Pseudostem base 0.0 29.5 30.8 31.8 b a a a Root 0.0 49.6 53.4 54.8 c b ab a

A
Colonization (%)

For each plant part, gures followed by similar letters are not signicantly different at P > 0.0085 (Dunn Sidak correction factor).

100 90 80 70 60 50 40 30 20 10 0 1.5 10

1.5 10 1h

1.5 10 2h

1.5 10 4h

3.2. Plant growth At the beginning of the experiments, plant growth was signicantly different among experimental trials (F P 170.91, df = 2, P < 0.0001). For all parameters, plants used in trial 3 were better developed than those used in trials 1 and 2. However, for each trial, the plants were uniformly distributed with no signicant difference across treatments for all growth parameters (v2 = 9.21 or F 6 1.33, df = 15, P P 0.18). Irrespective of dipping duration or inoculum dose, plant growth was not affected by B. bassiana treatment at harvest (v2 = 13.19 or F 6 0.99, df = 15, P P 0.47) (Table 3). 4. Discussion Irrespective of dose or exposure duration, virtually all tissue cultured banana plants were colonized by B. bassiana following inoculation. However, percentage colonization varied among plant parts, with greater colonization occurring in banana roots and rhizomes than pseudostem bases. These ndings reect those of an earlier study (Akello et al., 2007), who found that pseudostem base colonization (27.140.9%) was lower than rhizome (45.678.7%) and root (40.068.3%) colonization, at 1 month after inoculation. Differential B. bassiana colonization of plant parts was equally demonstrated in corn (Zea mays L.) and cocoa (Theobroma cacao L.) by Bing and Lewis (1991) and Posada and Vega (2005), respectively. In corn, the fungus was most frequently isolated from the internode below the primary ear and less frequently from the leaf collar at the primary ear. In cocoa, colonization rates in roots were higher than those in stems and leaves. In the current study, roots and rhizomes may have been more successfully colonized than pseudostem bases because roots and rhizomes came into direct contact with the conidial suspension at the time of inoculation. Consequently, these plant parts might have acted as initial entry routes for B. bassiana into the plants and hence were colonized rst by the fungus, prior to its spread into the pseudostem base. The optimal dose and exposure duration differed depending on plant part. Though variable, colonization of roots, rhizomes and pseudostem bases was primarily affected by inoculum dose and, in general, increased with increasing dose up to 1.5 107 conidia/ml, but decreased above this dose. Most likely, increasing inoculum dose increased the number of viable conidia that attached on the banana roots and rhizomes, which in turn promoted higher fungal infectivity and colonization of the plants. However, in some instances, colonization at a particular dose depended on exposure duration, indicating that for some doses improved colonization was attained when plants were dipped for a specic period of time. Under the conditions of the study, B. bassiana failed to prove detrimental to plant growth. It would appear therefore that B. bassiana is neither phytotoxic nor pathogenic to tissue cultured banana plants at levels which provide maximum plant colonization or even at the highest dose or longest exposure duration. Previously, endophytic B. bassiana was found to cause no adverse effects when articially introduced into corn (Bing and Lewis, 1991; Lewis et al.,

Inoculum dose (conidia/ml)

B
Colonization (%)

100 90 80 70 60 50 40 30 20 10 0 0 1.5 105 1.5 10


1h

1.5 10

1.5 10
4h

Inoculum dose (conidia/ml)


2h

C
Colonization (%)

100 90 80 70 60 50 40 30 20 10 0 0

1.5 10

1.5 10

1.5 10

1.5 10

Inoculum dose (conidia/ml)


1h 2h 4h

Fig. 3. The effect of dose and dipping duration on colonization of tissue-cultured banana (cv. Kibuzi, Musa sp. AAA-EA) plant rhizome, pseudostem base and root, 1 month after inoculating with Beauveria bassiana strain G41. Letters A, B and C denote rhizome, pseudostem base and root colonization, respectively. Colonization (%) represents the number of colonized segments divided by total number of cultured segments100.

2001) or banana (Akello et al., 2007) at doses of up to 2 1010 conidia/ml. Our study aimed to dene the optimal B. bassiana dose and dipping duration for maximizing colonization of banana plants. It is speculated that exposure to high doses for prolonged periods of time may increase colonization and persistence of endophytic B. bassiana in plant tissues and offer better protection against the banana weevil larvae. Our results show that B. bassiana dose of up to 1.5 108 conidia/ml does not affect banana growth, which provides the basis for more extensive determination of the pest management potential of B. bassiana as an endophyte in banana against banana weevils. Since an inoculum dose of 1.5 107 conidia/ml attained the highest rhizome colonization when plants were dipped for 2 h, this dose/time duration should be adopted, especially since

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J. Akello et al. / Biological Control 49 (2009) 610

Table 3 Growth of tissue cultured banana (Musa sp., AAA-EA, cv. Kibuzi) plants, 1 month after dipping in Beauveria bassiana suspensions of 1.5 105, 1.5 106, 1.5 107 and 1.5 108 conidia/ml for 1, 2 and 4 h, or dipping plants in 0.01% Tween 80 Parameter Exp. 1 Day 0 Plant height (cm)a Leaf length (cm)b Leaf width (cm)c Number of standing leaves Fresh weight (g)d Dry weight (g)e 5.7 0.1 12.2 0.2 4.9 0.1 3.2 0.0 Day 30 8.3 0.2 16.0 0.2 7.1 0.1 5.3 0.1 10.2 0.3 1.1 0.0 Exp. 2 Day 0 8.5 0.1 15.6 0.2 6.7 0.1 4.4 0.1 Day 30 9.6 0.2 19.1 0.2 8.7 0.1 5.5 0.1 17.2 0.5 1.7 0.1 Exp. 3 Day 0 11.7 0.2 18.5 0.3 8.4 0.5 4.9 0.1 Day 30 19.0 0.2 26.7 0.3 12.5 0.1 5.6 0.1 40.2 0.7 3.8 0.1

Since growth was not signicantly different across treatments, all data within a single experiment were combined. Day 0 and 30 represent measurements taken on the day of plant inoculation and harvest, respectively. For both days, growth was not signicantly different across treatments (P < 0.05). a The distance from the soil level to the youngest leaf axil. b The distance from the leaf apex to the leaf stalk of the youngest leaf. c Width at the widest part of the lamina of the youngest leaf. d Fresh shoot (pseudostem together with leaves) weight. e Dry shoot weight.

the targeted banana weevil larvae primarily affect the rhizome. Prolonging the dipping duration and/or increasing the inoculum dose appear not to improve colonization. Acknowledgments This project was made possible through nancial support from the Bundesministerium fr Wirtschaftliche Zusammenarbeit (BMZ), Germany, in the Managing Micro-organisms to Enhance Plant Health for Sustainable Banana Production in Eastern Africa project. We thank Dr. Caroline Nankinga of Kawanda Agricultural Research Institute (KARI), Uganda, for providing the B. bassiana strain G41 and the staff of the International Institute of Tropical Agriculture (IITA), Uganda, for technical assistance. References
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