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Effects of plant-growth-promoting microorganisms and fertilizers on growth of cabbage and tomato and Spodoptera litura performance
Effects of plant-growth-promoting microorganisms and fertilizers on growth of cabbage and tomato and Spodoptera litura performance Yuwatida Sripontan, Ching-Wen Tan, Mei-Hua Hung, Chiu-Chung Young, Shaw-Yhi Hwang PII: DOI: Reference:
S1226-8615(14)00067-3 doi: 10.1016/j.aspen.2014.05.007 ASPEN 537
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
9 December 2013 13 May 2014 15 May 2014
Please cite this article as: Sripontan, Yuwatida, Tan, Ching-Wen, Hung, Mei-Hua, Young, Chiu-Chung, Hwang, Shaw-Yhi, Effects of plant-growth-promoting microorganisms and fertilizers on growth of cabbage and tomato and Spodoptera litura performance, Journal of Asia-Pacific Entomology (2014), doi: 10.1016/j.aspen.2014.05.007
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ACCEPTED MANUSCRIPT Effects of plant-growth-promoting microorganisms and fertilizers on growth of cabbage
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and tomato and Spodoptera litura performance
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Yuwatida Sripontana, Ching-Wen Tana, Mei-Hua Hungb, Chiu-Chung Youngb, Shaw-Yhi Hwanga,* Department of Entomology, College of Agriculture and Natural Resources, National Chung Hsing
University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.
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Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources,
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National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.
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ACCEPTED MANUSCRIPT ABSTRACT Fertilizer and plant-growth-promoting microorganisms (PGPMs) both benefit crop growth;
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however, little is known about the interaction effects when they are combined. This study
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assessed the effect of PGPMs and fertilizer on plant growth, foliar chemistry, and subsequent
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insect feeding. Cabbage and tomato plants were inoculated with PGPMs (fungi and bacteria) and various levels of fertilization. Plant growth parameters (fresh weight, dry weight, and leaf
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area) and foliar chemistry (water content, protein content, and polyphenol oxidase activity) were then analyzed. In addition, foliage was also fed to the third instar larvae of Spodoptera
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litura to evaluate foliage quality. The results indicated that plant performance differed significantly among treatments, and the combined fungi Meyerozyma guilliermondii and
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fertilizer treatment promoted the greatest plant growth. In summary, PGPMs and fertilization
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can have their own effect; their interaction effect, however, need to be clarified.
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Keywords: plant-growth-promoting microorganisms (PGPMs), fertilizer, foliar chemistry,
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insect performance
* Corresponding author: Tel.: +886 422840363; fax: +886 4 22875024. E-mail address: [email protected] (S.-Y. Hwang).
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ACCEPTED MANUSCRIPT Introduction
Nearly all plant species undergo various levels of herbivory during their life spans and
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have developed various strategies to oppose these attacks (Karban and Baldwin, 1997;
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Harrison, 2005; Johnson and Agrawal, 2005). They protect themselves against herbivory by
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using a set of morphological and chemical defense strategies (Karban and Baldwin, 1997; Dicke and Hilker, 2003; Harrison, 2005; Johnson and Agrawal, 2005). Chemical defense strategies involve secondary metabolites and proteins that may be present constitutively or
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induced by challenges such as herbivore wounding (Ryan, 1990; Bennett and Wallsgrove,
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1994; Duffey and Stout, 1996; Zhu-Salzman et al., 2008). Although the evolution of such defense traits can be genetically fixed (Berenbaum et al., 1986; Alder et al., 1995; Hwang and Lindroth, 1997), the outcome of such traits might also be amended by other environmental
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factors (Bryant et al., 1983; Herms and Mattson, 1992). Various environmental features are considered to affect plants’ allocation of resources to
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defensive compounds (Bryant et al., 1983; Herms and Mattson, 1992). Nutrient accessibility has been considered a vital factor that influences plant growth and the distribution of limited
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resources (Bryant et al., 1983; Hemming and Lindroth, 1999). Fertilization has been considered a fundamental method of improving soil nutrient availability for plants and may consequently affect the growth, time of maturity, plant part size, and phytochemical content of plants (Myers, 1985; Hemming and Lindroth, 1999; Altieri and Nicholls, 2003; MeviSchütz et al., 2003). The phytochemical changes caused by fertilization in host plants may successively affect the pest species that use the host plants (Altieri and Nicholls, 2003). Microbial cooperation in the rhizosphere is essential for the sustainability of soil fertilizer and plant growth. Previous literature has indicated that mutualistic microbials in soil improve nutrient availability for plants and provide additional benefits, such as more efficient 3
ACCEPTED MANUSCRIPT uptake of minerals and water, disease resistance, protection from heavy metal toxicity, and an improved soil structure (Clark and Zeto, 1996; Pawlowska et al., 2000; Nichols and Wright,
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2004). Plant-growth-promoting microorganisms (PGPMs) are defined as soil-borne bacteria
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and fungi with plant promotion or protection activities (Bashan and de-Bashan, 2005;
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Shwetha et al., 2008). PGPMs have been reported to affect plants’ yield, physiology, growth and germination rates, and protein, mineral, and chlorophyll content (Glick, 2004; Bashan and de-Bashan, 2005; Lai et al., 2008). In general, microorganisms promote plant growth in 2
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processes: (1) PGPMs might fix atmospheric nitrogen to facilitate the absorption of
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solubilizing phosphorus and iron as well as to elevate the production of plant hormones, such as auxins, gibberellins, and cytokinins; and (2) PGPMs might also induce plants’ resistance to
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phytopathogens (bacteria, fungi, and viruses) (Bashan and de-Bashan, 2005; Figueiredo et al.,
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2011), insect pests (Zehnder et al., 1997; Ramamoorthy et al., 2001), and nematode pests (Sikora, 1988; Ramamoorthy et al., 2001). Therefore, using selected PGPMs to promote host
practices.
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plants’ resistance to insect pests maybe a potential alternative for sustainable agricultural
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For example, Zehnder et al. (1997) determined that applying plant-growth-promoting rhizobacteria (PGPR) can induce resistance against cucumber beetle feeding in cucumbers. The cotton inoculated with Pseudomonas gladiolican also reduced the relative growth rate and digestibility of Helicoverpa armigera because of the induced increase in foliar polyphenol and terpenoid content (Qingwen et al., 1998). In addition, one study indicated that, when rice (Oryza sativa) was inoculated with various PGPR strains, the incidence of rice leaf folder (Cnaphalocrocis medinalis) was reduced (Loganathan et al., 2010) because of a greater accumulation of enzyme lipoxygenase and chitinase activity against leaf folder insects. Therefore, numerous beneficial soil-borne microbes are suggested to cooperate with
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ACCEPTED MANUSCRIPT plants to combat insect herbivores by promoting plant growth and inducing resistance (Pineda et al., 2010).
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Thus, PGPMs may play particular roles in a plant’s induced defense response. However,
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such induced responses may vary among abiotic-ecological conditions (Stout et al., 1998; van
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Dam et al., 2001). Among these environmental factors, the extent of resource availability was observed to exert a profound impact on the level of the induced defense response (Borowicz
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et al., 2003; Barto et al., 2008) because several defensive substances contained elements derived from nutrients (Herms and Mattson, 1992). The effect of the interaction between
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nutrient availability and microbial-mediated induction response has not previously been addressed and the effect of the interaction-induced responses on subsequent herbivore
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performance also remains unknown. Therefore, this study assessed the interaction effects of
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resource availability and PGPMs on plant growth performance and induced response as well
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as on the performance of Spodoptera litura.
Plants
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Materials and methods
Two plant species, cabbage (Brassica oleracea L. var. capitata L.) and tomato (Lycopersican esculentum Mill. Var. cerasiforme (Dunal) Alef.), were used in the experiment. Seeds were sown with soil (Stender peat substrate, Known-You Seed Co., Ltd., Taiwan) in 104-well plates in a greenhouse (25±2 °C, 16:8 h light:dark photoperiod) and watered daily. After 2 wk, the seedlings were transplanted into 12.7 cm pots filled with field soil. Before planting, the chemical properties of the soil were analyzed by the Soil Survey and Testing Center, National Chung Hsing University, Taichung, Taiwan. Table 1 shows the 5
ACCEPTED MANUSCRIPT test results. Forty-seven-day-old cabbage and 51-d-old tomato plants were used for the
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bioassays.
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Insect
The eggs of Spodoptera litura were collected from a field in Taichung city, Taiwan
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and kept in 250-mL plastic rearing cups, and small wet cotton balls were added to provide moisture. The rearing cups were stored in a growth chamber (25°C, 16:8 h light:dark
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photoperiod; S.I.C. Co., Taiwan,). After the eggs hatched, the larvae were also reared in the 250-mLplastic rearing cups and fed an artificial diet (Yadav et al., 2010). Pupa were
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collected, separated according to sex, and placed in the 250-mL plastic rearing cups. After
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eclosion, the adults were paired (10 pairs) in a plastic cylinder (21 cm height × 14.9 cm diameter) and tissue paper was used to cover the inside for adults to oviposit eggs. The plastic
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cylinders were placed under laboratory conditions and the adult moths were fed a sugar
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solution (Yadav et al., 2010). The third instars of the insects were used for the bioassay.
Microbials and Fertilizer Treatments To evaluate the effects of fertilization, microbials, and their interactions on plants and herbivores, 3 fertilization levels (zero, half, and full) and 3 groups of microorganisms (control, fungi, and bacteria) were used in this study. The fungi treatment consisted of only one fungi species, Meyerozyma guilliermondii. Two types of bacteria mixture were used for the bacterial treatment. The first type of bacterial mixture was labeled B1 and comprised 3 bacterial species: Burkholderia phytofirmans, Rhizobium miluonense, and Rhizobium lusitanum. The second type of bacterial mixture (B2) contained 2 species: Bacillus subtilis 6
ACCEPTED MANUSCRIPT and Ochrobactrum pseudogrignonense (Table 2). Each microbial used in this study was observed to improve plants’ ability to uptake nutrients and minerals (Hung et al., 2005;
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Rekha et al., 2007; Nakayan et al., 2013). Therefore, 9 treatments were applied in this study:
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(1) no fertilization and no microbials, (2) half fertilization level and no microbials, (3) full
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fertilization level and no microbials, (4) fungi only, (5) half fertilization level and fungi, (6) full fertilization level and fungi, (7) bacterial mixtures, (8) half fertilization level and bacterial mixtures, and (9) full fertilization level and bacteria. Each microorganism used in
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this study was provided by the Laboratory of Soil Environmental Microbiology and
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Biochemistry, Department of Soil and Environmental Science, National Chung Hsing University. After the seeds were sown for 2 wk, the microbial suspension [F suspension
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(>108cfu mL-1), B1 suspension (>108cfu mL-1), and B2 suspension (≥108cfu mL-1)] were
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diluted 200 times using distilled water and then poured into plastic trays. The seedlings in the 104-well plates were soaked in the microbial suspension for 15 min as the first inoculation.
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After 2 d, the seedlings were transplanted into 12.7 cm pots filled with field soil. Every week after the transplant, 50 mL of various microbial solutions were added into each pot, and 50
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mL of water were added to plants in the control treatments. Five microbial inoculations were conducted in this study, and the concentration was increased gradually to promote their effects. The microbials were diluted 100 times using distilled water in the second and third inoculations, and were diluted 50 times using distilled water in the fourth and fifth inoculations (Table 3). In this study, we applied 5 time and different concentration of PGPMs to the soil. The amount of PGPMs would decline through time; therefore, we would like to make sure that the soil contained the PGPMs. At the first week’s inoculation, the plants were small and only low dose of PGPMs was applied. After the settle period, the plants grew well and had lots of roots, increased concentration of PGPMs was then used. Regarding the fertilizer treatments, a commercial synthetic fertilizer (20-20-20 N-P-K, Hyponex® 4; 7
ACCEPTED MANUSCRIPT Hyponex Co., Marysville, OH, USA) was used. The fertilizer was first dissolved in water and then added to the soil. Plants were given 50 mL of water, half of the recommended
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concentration (0.5 g/1000 mL), or the recommended concentration (1 g/1000 mL) for none,
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half-level, and full-level treatments, respectively. The fertilizer was treated weekly. The
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foliage of the cabbage (47 d after sowing) and tomato (51 d after sowing) plants were collected for plant performance analysis. The foliar water content, leaf area, and biomass of
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aboveground parts were measured as indicators of plant growth performance.
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Insect Performance Bioassay
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The insect performance bioassay was conducted to evaluate the effect of differently
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treated foliage on the relative growth rate of S. litura. The seventh leaf of cabbage plants and the fourth leaf of tomato plants were removed from the base of the plant by using surgical
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scissors and placed individually into petri dishes (9 cm in diameter). The petioles of the leaves were kept in a 2-mL Eppendorftube with RO water to maintain freshness. Third instar
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S. litura larvae were then weighed and individually placed on differently treated leaves (25 °C, 16:8 h light:dark photoperiod). The larvae were allowed to feed on the foliage for 48 h. They were subsequently separated, frozen, oven-dried, and weighed. Ten replications (larvae) were conducted for each treatment. At the same time of the bioassay, fresh weights of 15 third instar larvae were measured individually and then oven-dried at 45 °C. After 1 wk, the dry weights of the larvae were measured again. The average water content of the larvae was used to calculate the initial larval dry weight used in the feeding study. The relative growth rate (RGR) was calculated using the following equation: ((final dry weight of insect-initial dry weight of insect)/initial dry weight of insect)/duration) (Waldbauer, 1968; Farrar et al.,
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ACCEPTED MANUSCRIPT 1989; Schoonhoven et al., 1998). RGR was used as an indicator of insect growth
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performance.
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Foliar Chemistry
In this study, we measured foliar protein content and polyphenol oxidase activity. The
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seventh leaf of the cabbage plant and the fourth leaf of the tomato plant were collected during the bioassay for foliar chemical analysis. Five plants were used for each plant species. Leaf
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samples were ground using liquid nitrogen and then homogenized in a 7% grinding buffer (polyvinylpolypyrrolidone in a potassium phosphate buffer, pH 7). Leaf ground extract (1
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mL) was mixed with 100 µL of 10% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in a
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microtube. The crude extract solution was then centrifuged at 4 °C at 10 000 rpm for 15 min, after which the resulting supernatant was used for determining enzyme activity. To quantify
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the amount of protein, a standard curve was prepared using bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) (Bradford, 1976; Tan et al., 2011; Tan et al., 2012).
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Polyphenol oxidase activity was measured based on the procedures of Stout et al. (1999) to calculate the formation rate of melanin-like material from catechol. For this assay, 15 µL of a supernatant liquid was mixed with catechol (0.1 M potassium phosphate buffer, pH 8). After mixing for 1 min, an absorbance value below 470 nm was recorded (Ryan et al., 1982; Thaler et al., 1996; Thaler et al., 2001; Cipollini et al., 2004).
Statistical Analysis
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ACCEPTED MANUSCRIPT Mean and standard error values were calculated for plant growth, insect performance, and foliar protein and polyphenol oxidase content. Two factors of this study included PGPM
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and fertilizer. A two-way analysis of variance (ANOVA) and Tukey multiple-range test
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(Version 6.2; SAS Institute Inc., Cary, NC, USA, 1996) were used for comparing the
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interaction effects between fertilization and microbial application.
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Results
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Plant Growth Performance
The effects of microbial application and fertilization on plant growth performance
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(foliar fresh and dry weight and leaf area) differed. The results indicated that the fresh weight
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of cabbage differed significantly among various microbial treatments (F= 35.98, P=0.0001);
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fungi treatment exhibited the most significant effect on foliar fresh weight (Fig. 1). In addition, cabbage fresh weight increased markedly as the level of fertilization increased (F= 560.33, P=0.0001). Full-level fertilizer treatment produced a dry weight 2 times greater than
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that of the control treatment. Moreover, combined microbial and fertilizer application significantly affected the fresh weight of the cabbage (F= 4.94, P=0.0028). The results also indicated that the foliar dry weight of the cabbage differed significantly between the microbial (F= 18.76, P=0.0001) and fertilizer (F= 87.80, P=0.0001) treatments. In addition, a significant interaction effect was observed between microbial and fertilizer application regarding the foliar dry weight (F= 8.00, P=0.0001) (Fig. 1). Regarding the leaf areas, both microbial (F= 47.53, P=0.0001) and fertilizer (F= 342.50, P=0.0001) treatments exhibited profound effects. Fungi treatment using the full level of fertilization yielded the largest leaf area (Fig. 1). Additionally, combined microbial and fertilizer application significantly 10
ACCEPTED MANUSCRIPT affected the leaf areas (F= 4.70, P=0.0037). The results indicated that the fresh weight of the tomato did not differ among the
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microbial treatments (F= 1.40, P=0.2600). By contrast, nutrient availability significantly
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affected the fresh weight (F= 159.83, P=0.0001). Full fertilization treatment doubled the
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foliar fresh weight (Fig. 2). Moreover, the effects of microbial and fertilizer application on foliar fresh weight were not significant (F= 1.22, P=0.3195). The dry weight of the tomato
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was significantly altered by both microbial (F= 4.51, P=0.0178) and fertilizer application (F= 84.78, P=0.0001) (Fig. 2). However, the effects of microbial and fertilizer application on the
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foliar dry weight were independent of each other (no significant interaction) (F= 2.15, P=0.0944). The results indicated that the leaf area of the tomato was not affected by
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microbial treatment (F= 2.92, P=0.0670), whereas the level of fertilization significantly
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influenced the leaf area (F= 151.19, P=0.0001) (Fig. 2). In addition, the combined effect of
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Foliar Chemistry
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microbial and fertilizer application on the leaf area was not significant (F= 0.33, P=0.8532).
The effects of microbial and nutrient availability on nutrients (water and protein) and defense-related compounds (polyphenol oxidase, PPO) differed among treatments. Regarding the cabbage, foliar water content was observed to vary significantly among the microbial treatments (F= 25.77, P=0.0001), and overall, the fungi-treated cabbage exhibited greater water content (Fig. 3). In addition, fertilization slightly affected the foliar water content (F= 28.15, P=0.0001). Similarly, a significant interaction effect also occurred between microbial and fertilizer application regarding the foliar water content (F= 10.59, P=0.0001). The results indicated that protein content did not differ significantly among the microbial treatments (F= 11
ACCEPTED MANUSCRIPT 1.77, P=0.1851), whereas fertilizer application significantly affected the protein content (F= 7.62, P=0.0017) (Fig. 3). Moreover, the effect of microbial and fertilizer application on the
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cabbage’s protein content was nonsignificant (F= 1.51, P=0.2200). Microbial treatment did
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not affect the PPO activity (F= 3.39, P=0.0447), and the PPO activity also did not differ
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significantly among fertilizer treatments (F= 3.86, P=0.0302) (Fig. 3). However, the interaction between microbial and fertilizer application significantly influenced the PPO
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activity (F= 6.29, P=0.0006).
Regarding the tomato, the results indicated that microbial application (F = 4.82, P =
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0.0140) and the fertilization level (F= 26.74, P= 0.0001) significantly influenced the water content (Fig. 4). Moreover, the interaction between microbial and fertilizer application on the
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water content was not significant (F= 2.72, P=0.0446). Protein content did not differ
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significantly among the microbial treatments (F= 0.61, P=0.5515) (Fig. 4); however, the fertilization level significantly affected the protein content (F= 23.00, P=0.0001). The
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fertilization treatment contained a higher protein content than did the control treatment. However, the effect of the combined microbial and fertilizer treatment on the protein content
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was nonsignificant (F= 1.02, P=0.4093). The results revealed that PPO activity was not affected by microbial application (F= 4.64, P=0.0161) (Fig. 4), whereas the fertilization level significantly affected the PPO activity (F= 11.76, P=0.0001). Moreover, the effect of microbial and fertilizer application on PPO activity also did not differ significantly (F= 1.82, P=0.1463).
Insect Performance The results indicated that microbial and fertilizer application produced various effects 12
ACCEPTED MANUSCRIPT on the performance of S. litura larvae. Regarding the larvae that fed on the cabbage, the results indicated that microbial (F=4.03, P=0.0214) and fertilizer (F= 5.92, P=0.0040)
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application significantly affected the RGR (Fig. 5). Moreover, the interaction effect of
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microbial and fertilizer application on the performance of S. litura was significant (F= 4.31,
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P=0.0033).
Regarding the larvae that fed on the tomato foliage, the results indicated that the RGR
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was not significantly influenced by microbial treatment (F= 1.93, P=0.1511) (Fig. 5). However, the fertilization level significantly affected the performance of S. litura (F= 22.15,
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P=0.0001). The larvae grew 2 times faster on the foliage when using the full fertilizer treatment than when using the control treatment. In addition, the interaction between
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Discussion
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microbial and fertilizer application on RGR was significant (F= 3.08, P=0.0207).
The results of this study indicate that microbial application and fertilizer application
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produce significant and different effects on plant growth and insect performance. In addition, microbial treatment can promote plant growth and significantly reduce the amount of fertilizer applied.
Fertilizer may be the most crucial factor that affects plant growth and yield. Fertilizer application can promote plant growth. A previous study indicated that fertilized plants contain leaf numbers and plant biomass 5 times higher than those of unfertilized plants (Hsu et al., 2009). However, high level of fertilization may also lead to high insect damage levels. In addition to fertilizer, PGPMs in the soil environment have also been identified to play vital roles in plant growth. Studies have revealed that several species of PGPMs, such as Bacillus 13
ACCEPTED MANUSCRIPT sp. and Burkholderia vietnamiensis els., can significantly increase plant growth performance in numerous plant species (Kokalis-Burelle et al., 2002; Rekha et al., 2007). Fertilizer and
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PGPMs may independently facilitate plant growth and a combined effect may occur when
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they are used together. A previous study determined that combined PGPM and low fertilizer
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application produces plant biomass and nutrient uptake levels similar to those of full fertilizer treatment (Afzal and Bano, 2008; Adesemoye and Kloepper, 2009; Nakayan et al., 2013). Our results also indicated that microbials and fertilizer significantly influence plant growth.
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Moreover, we observed that plants treated with the fungi Meyerozyma guilliermondii and a
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half level of fertilization generate a plant biomass similar to that generated when using full fertilizer treatment, suggesting that adding these microbials can reduce the amount of
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fertilizer required and, consequently, reduce farming costs.
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Fertilizer and PGPM application may affect plants’ chemistry (Stout et al., 1998; Lucy et al., 2004; Hsu et al., 2009; Peric et al., 2009). Fertilizer normally exhibits a
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significant influence on the primary plant metabolites. Previous studies have indicated that high nutrient availability increases foliar protein and nutrient content (Stout et al., 1998;
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Prudic et al., 2005; Peric et al., 2009; Bybordi and Ebrahimian, 2013). The results of the current study also suggested that full fertilizer treatment produces twice the protein content. In addition, the levels of secondary plant metabolites, such as defensive proteins (trypsin inhibitor (TI) and PPO) and phenolics, can also be elevated by fertilization; this maybe due to an increase in resource availability (Stout et al., 1998; Hsu et al., 2009; Peric et al., 2009). The PPO activity of the tomato plant, however, decreased when applying fertilizer in this study, and the cause remains unclear. Moreover, because of their characteristic of enhancing root absorbing efficiency, PGPMs have been considered to influence the nutrient uptake of host plants and to increase the macro- and micronutrient and protein content of plants (Egamberdiyeva and Höflich, 2003; Glick, 2004; Bashan and de-Bashan, 2005; Lai et al., 14
ACCEPTED MANUSCRIPT 2008). PGPMs have been observed to increase the nitrogen content and mineral content of various plant species (Boddey and Dobereiner, 1988; Belimov and Dietz, 2000; Cakmakci et
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al., 2001; Dobbelaere et al., 2001; Probanza et al., 2002). Similarly, relevant literature has
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also indicated that PGPMs induce the production certain secondary plant metabolites
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(Figueiredo et al., 2011; Shanmugam and Kanoujia, 2011). Previous studies have determined that plants treated with PGPR increase the activity of peroxidase, PPO, phenylalanine ammonia-lyase, and proteinase inhibitors (Chen et al., 2000; Figueiredo et al., 2011;
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Shanmugam and Kanoujia, 2011). However, little is known about the interaction between the
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2 factors; only a few studies have indicated that the effect of PGPMs and fertilizer increases the concentrations of macronutrients (N, P, K, and Ca) and micronutrient (Mg, Fe, Zn, Cu,
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and Mn) in plants (Lai et al., 2008; Nakayan et al., 2013). Our results suggest that combined
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microbial and fertilizer treatment significantly increases foliar protein content. However, the PPO activity decreased during combined treatment. Therefore, the effect of PGPMs and
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fertilizer on the nutrient content of plants can be confirmed; however, the effects on plant defensive compounds remain unverified. Additional studies are required to clarify the effect
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and the mechanisms of PGPMs on the nutrient allocation of plants. Fertilizer and PGPMs are 2 vital factors that influence a plant’s chemistry and subsequently affect herbivorous insects’ performance (Ramamoorthy et al., 2001; Herm, 2002; Pineda et al., 2010). Previous studies have indicated that fertilized plants produce small amounts of defensive chemicals; thus, herbivorous insects perform better on fertilized plants than on unfertilized plants (Altieri and Nicolls, 2003; Prudic et al., 2005; Hsu et al., 2009). PGPMs promote plant growth by increasing root absorption; however, relevant literature has reported that PGPMs may also induce a plant’ defense responses (Ramamoorthy et al., 2001; Pineda et al., 2010). For example, a previous study revealed that PGPR-treated plants are resistant to feeding by the cucumber beetle Diabrotica undecimpunctata howardi Barber 15
ACCEPTED MANUSCRIPT (Zehnder et al., 1997; Zehnder et al., 2001), leaf folder Cnaphalocrocis medinalis (Saravanakumar et al., 2007), and leaf miner Aproaerema modicella (Senthilraja et al., 2010).
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Fertilization and PGPMs exhibit varied effects on plants and, consequently, on herbivorous
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insects; however, the combined PGPR and fertilizer effect on insect performance is largely
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unknown. Our results indicate that microbial application and fertilization could significantly affect the RGR of the third instar larvae of S. litura; but the interaction effect is still not clear. In summary, our study suggests that PGPMs and fertilizer exert various influences on
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plant growth performance, foliar chemistry, and insect performance. Both treatments exhibit
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positive effects on plant performance. However, the interaction effect of the microbials and fertilizer on plants remains unclear. Therefore, additional studies are required to clarify the
Acknowledgments
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interaction effect when using them together in promoting crop production and pest control.
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We thank Wallace Academic Editing and two anonymous reviewers for their
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comments on the manuscript.
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ACCEPTED MANUSCRIPT Phosphate-solubilizing soil yeast Meyerozyma guilliermondii CC1 improves maize (Zea may L.) productivity and minimizes requisite chemical fertilization. Plant Soil.
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Sikora, R.A., 1988. Interrelationship between plant health promoting rhizobacteria, plant
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parasitic nematodes and soil microorganisms. Med. Fac. Landbouww. Rijksuniv.
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Stout, M.J., Fidantsef, A.L., Duffey, S.S., Bostock, R.M., 1999. Signal interactions in pathogen and insect attack: systemic plant mediated interactions between pathogens
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ACCEPTED MANUSCRIPT protease inhibitor response in Nicotiana attenuate. J. Chem. Ecol. 27, 547-568. Waldbauer, G.P., 1968. The consumption and utilization of food by insects. Adv. Insect
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Physiol. 5, 229-288.
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litura. Environ. Entomol. 39, 1990-1996.
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radish (Raphanus sativus) plants and their effects on performance of Spodoptera
Zehnder, G., Kloepper, J., Changbin, Y., Wei, G., 1997. Induction of systemic resistance in
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cucumber against cucumber beetles (Coleoptera: Crysomelidae) by Plant-Growth-
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Promoting-Rhizobacteria. J. Econ. Entomol. 90, 391-396. Zehnder, G.W., Murphy, J.F., Sikora, E.J., Kloepper, J.W., 2001. Application of rhizobacteria
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Spectrum Defenses against Multiple Herbivores. Plant Physiol. 146, 852-858.
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ACCEPTED MANUSCRIPT Table 1 Chemical properties of the field soil used in the study.
6.71
EC (µS/cm)
30.9
OM (%)
0.34
Total N (g kg-1)
0.595
P (mg kg-1)
76.13404
K (mg kg-1)
35.75068
Ca (mg kg-1)
443.2
Mg (mg kg-1)
148.3595
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592.1538
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Cu (mg kg-1)
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Fe (mg kg-1) Mn (mg kg-1)
Zn (mg kg-1)
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pH
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Property Value ________________________________________________
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________________________________________________
222.9418 6.513184 45.12512
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______________________________________________
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ACCEPTED MANUSCRIPT Table 2 The microorganisms used for the study.
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B1a
Bacteria (B)
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Rhizobium miluonense Rhizobium lusitanum
B2a
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Bacillus subtilis
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Burkholderia phytofirmans
Ochrobactrum pseudogrignonense
Fungi (F)
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B1 and B2, mixture of the bacterial strains.
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a
Meyerozyma guilliermondii
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ACCEPTED MANUSCRIPT Table 3 Microbial mixtures application process used for the PGPMs treatments. _____________________________________________________________ Fungi
Bacteria
Concentration
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Inoculation
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(PGPMs: water)
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_____________________________________________________________ Fa
B1b
2nd inoculation
F
B2b
3rd inoculation
F
B1
4th inoculation
F
B2
1:50
5th inoculation
F
B1
1:50
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1st inoculation
1:200 1:100 1:100
___________________________________________________________
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F, fungi. b B1 and B2, different bacterial mixtures.
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a
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Fig. 1. Growth performance of cabbage plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test)
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Fig. 2. Growth performance of tomato plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test)
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Fig. 3. Chemical components of cabbage plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test) 30
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Fig. 4. Chemical components of tomato plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test) 31
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B
Fig. 5. Relative growth rate of third instar larva of Spodoptera litura on cabbage plants (A) and tomato plants (B) treated with microbials and fertilizer. Mean±SE (n = 10) (P < 0.05, Tukey’s test)
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ACCEPTED MANUSCRIPT Highlights
We assessed the effect of PGPMs and fertilizer on plant and insect feeding.
Cabbage and tomato plants were inoculated with PGPMs (fungi and bacteria) and
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We found fungi Meyerozyma guilliermondii and fertilizer treatment promoted the
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greatest plant growth.
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various levels of fertilization.
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S1226-8615(14)00067-3 doi: 10.1016/j.aspen.2014.05.007 ASPEN 537
To appear in:
Journal of Asia-Pacific Entomology
Received date: Revised date: Accepted date:
9 December 2013 13 May 2014 15 May 2014
Please cite this article as: Sripontan, Yuwatida, Tan, Ching-Wen, Hung, Mei-Hua, Young, Chiu-Chung, Hwang, Shaw-Yhi, Effects of plant-growth-promoting microorganisms and fertilizers on growth of cabbage and tomato and Spodoptera litura performance, Journal of Asia-Pacific Entomology (2014), doi: 10.1016/j.aspen.2014.05.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Effects of plant-growth-promoting microorganisms and fertilizers on growth of cabbage
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and tomato and Spodoptera litura performance
a
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Yuwatida Sripontana, Ching-Wen Tana, Mei-Hua Hungb, Chiu-Chung Youngb, Shaw-Yhi Hwanga,* Department of Entomology, College of Agriculture and Natural Resources, National Chung Hsing
University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.
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b
Department of Soil and Environmental Sciences, College of Agriculture and Natural Resources,
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National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwan.
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ACCEPTED MANUSCRIPT ABSTRACT Fertilizer and plant-growth-promoting microorganisms (PGPMs) both benefit crop growth;
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however, little is known about the interaction effects when they are combined. This study
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assessed the effect of PGPMs and fertilizer on plant growth, foliar chemistry, and subsequent
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insect feeding. Cabbage and tomato plants were inoculated with PGPMs (fungi and bacteria) and various levels of fertilization. Plant growth parameters (fresh weight, dry weight, and leaf
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area) and foliar chemistry (water content, protein content, and polyphenol oxidase activity) were then analyzed. In addition, foliage was also fed to the third instar larvae of Spodoptera
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litura to evaluate foliage quality. The results indicated that plant performance differed significantly among treatments, and the combined fungi Meyerozyma guilliermondii and
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fertilizer treatment promoted the greatest plant growth. In summary, PGPMs and fertilization
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can have their own effect; their interaction effect, however, need to be clarified.
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Keywords: plant-growth-promoting microorganisms (PGPMs), fertilizer, foliar chemistry,
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insect performance
* Corresponding author: Tel.: +886 422840363; fax: +886 4 22875024. E-mail address: [email protected] (S.-Y. Hwang).
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ACCEPTED MANUSCRIPT Introduction
Nearly all plant species undergo various levels of herbivory during their life spans and
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have developed various strategies to oppose these attacks (Karban and Baldwin, 1997;
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Harrison, 2005; Johnson and Agrawal, 2005). They protect themselves against herbivory by
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using a set of morphological and chemical defense strategies (Karban and Baldwin, 1997; Dicke and Hilker, 2003; Harrison, 2005; Johnson and Agrawal, 2005). Chemical defense strategies involve secondary metabolites and proteins that may be present constitutively or
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induced by challenges such as herbivore wounding (Ryan, 1990; Bennett and Wallsgrove,
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1994; Duffey and Stout, 1996; Zhu-Salzman et al., 2008). Although the evolution of such defense traits can be genetically fixed (Berenbaum et al., 1986; Alder et al., 1995; Hwang and Lindroth, 1997), the outcome of such traits might also be amended by other environmental
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factors (Bryant et al., 1983; Herms and Mattson, 1992). Various environmental features are considered to affect plants’ allocation of resources to
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defensive compounds (Bryant et al., 1983; Herms and Mattson, 1992). Nutrient accessibility has been considered a vital factor that influences plant growth and the distribution of limited
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resources (Bryant et al., 1983; Hemming and Lindroth, 1999). Fertilization has been considered a fundamental method of improving soil nutrient availability for plants and may consequently affect the growth, time of maturity, plant part size, and phytochemical content of plants (Myers, 1985; Hemming and Lindroth, 1999; Altieri and Nicholls, 2003; MeviSchütz et al., 2003). The phytochemical changes caused by fertilization in host plants may successively affect the pest species that use the host plants (Altieri and Nicholls, 2003). Microbial cooperation in the rhizosphere is essential for the sustainability of soil fertilizer and plant growth. Previous literature has indicated that mutualistic microbials in soil improve nutrient availability for plants and provide additional benefits, such as more efficient 3
ACCEPTED MANUSCRIPT uptake of minerals and water, disease resistance, protection from heavy metal toxicity, and an improved soil structure (Clark and Zeto, 1996; Pawlowska et al., 2000; Nichols and Wright,
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2004). Plant-growth-promoting microorganisms (PGPMs) are defined as soil-borne bacteria
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and fungi with plant promotion or protection activities (Bashan and de-Bashan, 2005;
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Shwetha et al., 2008). PGPMs have been reported to affect plants’ yield, physiology, growth and germination rates, and protein, mineral, and chlorophyll content (Glick, 2004; Bashan and de-Bashan, 2005; Lai et al., 2008). In general, microorganisms promote plant growth in 2
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processes: (1) PGPMs might fix atmospheric nitrogen to facilitate the absorption of
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solubilizing phosphorus and iron as well as to elevate the production of plant hormones, such as auxins, gibberellins, and cytokinins; and (2) PGPMs might also induce plants’ resistance to
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phytopathogens (bacteria, fungi, and viruses) (Bashan and de-Bashan, 2005; Figueiredo et al.,
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2011), insect pests (Zehnder et al., 1997; Ramamoorthy et al., 2001), and nematode pests (Sikora, 1988; Ramamoorthy et al., 2001). Therefore, using selected PGPMs to promote host
practices.
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plants’ resistance to insect pests maybe a potential alternative for sustainable agricultural
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For example, Zehnder et al. (1997) determined that applying plant-growth-promoting rhizobacteria (PGPR) can induce resistance against cucumber beetle feeding in cucumbers. The cotton inoculated with Pseudomonas gladiolican also reduced the relative growth rate and digestibility of Helicoverpa armigera because of the induced increase in foliar polyphenol and terpenoid content (Qingwen et al., 1998). In addition, one study indicated that, when rice (Oryza sativa) was inoculated with various PGPR strains, the incidence of rice leaf folder (Cnaphalocrocis medinalis) was reduced (Loganathan et al., 2010) because of a greater accumulation of enzyme lipoxygenase and chitinase activity against leaf folder insects. Therefore, numerous beneficial soil-borne microbes are suggested to cooperate with
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ACCEPTED MANUSCRIPT plants to combat insect herbivores by promoting plant growth and inducing resistance (Pineda et al., 2010).
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Thus, PGPMs may play particular roles in a plant’s induced defense response. However,
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such induced responses may vary among abiotic-ecological conditions (Stout et al., 1998; van
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Dam et al., 2001). Among these environmental factors, the extent of resource availability was observed to exert a profound impact on the level of the induced defense response (Borowicz
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et al., 2003; Barto et al., 2008) because several defensive substances contained elements derived from nutrients (Herms and Mattson, 1992). The effect of the interaction between
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nutrient availability and microbial-mediated induction response has not previously been addressed and the effect of the interaction-induced responses on subsequent herbivore
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performance also remains unknown. Therefore, this study assessed the interaction effects of
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resource availability and PGPMs on plant growth performance and induced response as well
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as on the performance of Spodoptera litura.
Plants
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Materials and methods
Two plant species, cabbage (Brassica oleracea L. var. capitata L.) and tomato (Lycopersican esculentum Mill. Var. cerasiforme (Dunal) Alef.), were used in the experiment. Seeds were sown with soil (Stender peat substrate, Known-You Seed Co., Ltd., Taiwan) in 104-well plates in a greenhouse (25±2 °C, 16:8 h light:dark photoperiod) and watered daily. After 2 wk, the seedlings were transplanted into 12.7 cm pots filled with field soil. Before planting, the chemical properties of the soil were analyzed by the Soil Survey and Testing Center, National Chung Hsing University, Taichung, Taiwan. Table 1 shows the 5
ACCEPTED MANUSCRIPT test results. Forty-seven-day-old cabbage and 51-d-old tomato plants were used for the
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bioassays.
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Insect
The eggs of Spodoptera litura were collected from a field in Taichung city, Taiwan
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and kept in 250-mL plastic rearing cups, and small wet cotton balls were added to provide moisture. The rearing cups were stored in a growth chamber (25°C, 16:8 h light:dark
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photoperiod; S.I.C. Co., Taiwan,). After the eggs hatched, the larvae were also reared in the 250-mLplastic rearing cups and fed an artificial diet (Yadav et al., 2010). Pupa were
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collected, separated according to sex, and placed in the 250-mL plastic rearing cups. After
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eclosion, the adults were paired (10 pairs) in a plastic cylinder (21 cm height × 14.9 cm diameter) and tissue paper was used to cover the inside for adults to oviposit eggs. The plastic
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cylinders were placed under laboratory conditions and the adult moths were fed a sugar
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solution (Yadav et al., 2010). The third instars of the insects were used for the bioassay.
Microbials and Fertilizer Treatments To evaluate the effects of fertilization, microbials, and their interactions on plants and herbivores, 3 fertilization levels (zero, half, and full) and 3 groups of microorganisms (control, fungi, and bacteria) were used in this study. The fungi treatment consisted of only one fungi species, Meyerozyma guilliermondii. Two types of bacteria mixture were used for the bacterial treatment. The first type of bacterial mixture was labeled B1 and comprised 3 bacterial species: Burkholderia phytofirmans, Rhizobium miluonense, and Rhizobium lusitanum. The second type of bacterial mixture (B2) contained 2 species: Bacillus subtilis 6
ACCEPTED MANUSCRIPT and Ochrobactrum pseudogrignonense (Table 2). Each microbial used in this study was observed to improve plants’ ability to uptake nutrients and minerals (Hung et al., 2005;
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Rekha et al., 2007; Nakayan et al., 2013). Therefore, 9 treatments were applied in this study:
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(1) no fertilization and no microbials, (2) half fertilization level and no microbials, (3) full
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fertilization level and no microbials, (4) fungi only, (5) half fertilization level and fungi, (6) full fertilization level and fungi, (7) bacterial mixtures, (8) half fertilization level and bacterial mixtures, and (9) full fertilization level and bacteria. Each microorganism used in
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this study was provided by the Laboratory of Soil Environmental Microbiology and
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Biochemistry, Department of Soil and Environmental Science, National Chung Hsing University. After the seeds were sown for 2 wk, the microbial suspension [F suspension
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(>108cfu mL-1), B1 suspension (>108cfu mL-1), and B2 suspension (≥108cfu mL-1)] were
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diluted 200 times using distilled water and then poured into plastic trays. The seedlings in the 104-well plates were soaked in the microbial suspension for 15 min as the first inoculation.
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After 2 d, the seedlings were transplanted into 12.7 cm pots filled with field soil. Every week after the transplant, 50 mL of various microbial solutions were added into each pot, and 50
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mL of water were added to plants in the control treatments. Five microbial inoculations were conducted in this study, and the concentration was increased gradually to promote their effects. The microbials were diluted 100 times using distilled water in the second and third inoculations, and were diluted 50 times using distilled water in the fourth and fifth inoculations (Table 3). In this study, we applied 5 time and different concentration of PGPMs to the soil. The amount of PGPMs would decline through time; therefore, we would like to make sure that the soil contained the PGPMs. At the first week’s inoculation, the plants were small and only low dose of PGPMs was applied. After the settle period, the plants grew well and had lots of roots, increased concentration of PGPMs was then used. Regarding the fertilizer treatments, a commercial synthetic fertilizer (20-20-20 N-P-K, Hyponex® 4; 7
ACCEPTED MANUSCRIPT Hyponex Co., Marysville, OH, USA) was used. The fertilizer was first dissolved in water and then added to the soil. Plants were given 50 mL of water, half of the recommended
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concentration (0.5 g/1000 mL), or the recommended concentration (1 g/1000 mL) for none,
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half-level, and full-level treatments, respectively. The fertilizer was treated weekly. The
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foliage of the cabbage (47 d after sowing) and tomato (51 d after sowing) plants were collected for plant performance analysis. The foliar water content, leaf area, and biomass of
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aboveground parts were measured as indicators of plant growth performance.
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Insect Performance Bioassay
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The insect performance bioassay was conducted to evaluate the effect of differently
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treated foliage on the relative growth rate of S. litura. The seventh leaf of cabbage plants and the fourth leaf of tomato plants were removed from the base of the plant by using surgical
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scissors and placed individually into petri dishes (9 cm in diameter). The petioles of the leaves were kept in a 2-mL Eppendorftube with RO water to maintain freshness. Third instar
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S. litura larvae were then weighed and individually placed on differently treated leaves (25 °C, 16:8 h light:dark photoperiod). The larvae were allowed to feed on the foliage for 48 h. They were subsequently separated, frozen, oven-dried, and weighed. Ten replications (larvae) were conducted for each treatment. At the same time of the bioassay, fresh weights of 15 third instar larvae were measured individually and then oven-dried at 45 °C. After 1 wk, the dry weights of the larvae were measured again. The average water content of the larvae was used to calculate the initial larval dry weight used in the feeding study. The relative growth rate (RGR) was calculated using the following equation: ((final dry weight of insect-initial dry weight of insect)/initial dry weight of insect)/duration) (Waldbauer, 1968; Farrar et al.,
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ACCEPTED MANUSCRIPT 1989; Schoonhoven et al., 1998). RGR was used as an indicator of insect growth
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performance.
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Foliar Chemistry
In this study, we measured foliar protein content and polyphenol oxidase activity. The
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seventh leaf of the cabbage plant and the fourth leaf of the tomato plant were collected during the bioassay for foliar chemical analysis. Five plants were used for each plant species. Leaf
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samples were ground using liquid nitrogen and then homogenized in a 7% grinding buffer (polyvinylpolypyrrolidone in a potassium phosphate buffer, pH 7). Leaf ground extract (1
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mL) was mixed with 100 µL of 10% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in a
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microtube. The crude extract solution was then centrifuged at 4 °C at 10 000 rpm for 15 min, after which the resulting supernatant was used for determining enzyme activity. To quantify
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the amount of protein, a standard curve was prepared using bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) (Bradford, 1976; Tan et al., 2011; Tan et al., 2012).
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Polyphenol oxidase activity was measured based on the procedures of Stout et al. (1999) to calculate the formation rate of melanin-like material from catechol. For this assay, 15 µL of a supernatant liquid was mixed with catechol (0.1 M potassium phosphate buffer, pH 8). After mixing for 1 min, an absorbance value below 470 nm was recorded (Ryan et al., 1982; Thaler et al., 1996; Thaler et al., 2001; Cipollini et al., 2004).
Statistical Analysis
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ACCEPTED MANUSCRIPT Mean and standard error values were calculated for plant growth, insect performance, and foliar protein and polyphenol oxidase content. Two factors of this study included PGPM
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and fertilizer. A two-way analysis of variance (ANOVA) and Tukey multiple-range test
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(Version 6.2; SAS Institute Inc., Cary, NC, USA, 1996) were used for comparing the
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interaction effects between fertilization and microbial application.
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Results
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Plant Growth Performance
The effects of microbial application and fertilization on plant growth performance
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(foliar fresh and dry weight and leaf area) differed. The results indicated that the fresh weight
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of cabbage differed significantly among various microbial treatments (F= 35.98, P=0.0001);
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fungi treatment exhibited the most significant effect on foliar fresh weight (Fig. 1). In addition, cabbage fresh weight increased markedly as the level of fertilization increased (F= 560.33, P=0.0001). Full-level fertilizer treatment produced a dry weight 2 times greater than
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that of the control treatment. Moreover, combined microbial and fertilizer application significantly affected the fresh weight of the cabbage (F= 4.94, P=0.0028). The results also indicated that the foliar dry weight of the cabbage differed significantly between the microbial (F= 18.76, P=0.0001) and fertilizer (F= 87.80, P=0.0001) treatments. In addition, a significant interaction effect was observed between microbial and fertilizer application regarding the foliar dry weight (F= 8.00, P=0.0001) (Fig. 1). Regarding the leaf areas, both microbial (F= 47.53, P=0.0001) and fertilizer (F= 342.50, P=0.0001) treatments exhibited profound effects. Fungi treatment using the full level of fertilization yielded the largest leaf area (Fig. 1). Additionally, combined microbial and fertilizer application significantly 10
ACCEPTED MANUSCRIPT affected the leaf areas (F= 4.70, P=0.0037). The results indicated that the fresh weight of the tomato did not differ among the
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microbial treatments (F= 1.40, P=0.2600). By contrast, nutrient availability significantly
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affected the fresh weight (F= 159.83, P=0.0001). Full fertilization treatment doubled the
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foliar fresh weight (Fig. 2). Moreover, the effects of microbial and fertilizer application on foliar fresh weight were not significant (F= 1.22, P=0.3195). The dry weight of the tomato
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was significantly altered by both microbial (F= 4.51, P=0.0178) and fertilizer application (F= 84.78, P=0.0001) (Fig. 2). However, the effects of microbial and fertilizer application on the
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foliar dry weight were independent of each other (no significant interaction) (F= 2.15, P=0.0944). The results indicated that the leaf area of the tomato was not affected by
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microbial treatment (F= 2.92, P=0.0670), whereas the level of fertilization significantly
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influenced the leaf area (F= 151.19, P=0.0001) (Fig. 2). In addition, the combined effect of
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Foliar Chemistry
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microbial and fertilizer application on the leaf area was not significant (F= 0.33, P=0.8532).
The effects of microbial and nutrient availability on nutrients (water and protein) and defense-related compounds (polyphenol oxidase, PPO) differed among treatments. Regarding the cabbage, foliar water content was observed to vary significantly among the microbial treatments (F= 25.77, P=0.0001), and overall, the fungi-treated cabbage exhibited greater water content (Fig. 3). In addition, fertilization slightly affected the foliar water content (F= 28.15, P=0.0001). Similarly, a significant interaction effect also occurred between microbial and fertilizer application regarding the foliar water content (F= 10.59, P=0.0001). The results indicated that protein content did not differ significantly among the microbial treatments (F= 11
ACCEPTED MANUSCRIPT 1.77, P=0.1851), whereas fertilizer application significantly affected the protein content (F= 7.62, P=0.0017) (Fig. 3). Moreover, the effect of microbial and fertilizer application on the
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cabbage’s protein content was nonsignificant (F= 1.51, P=0.2200). Microbial treatment did
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not affect the PPO activity (F= 3.39, P=0.0447), and the PPO activity also did not differ
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significantly among fertilizer treatments (F= 3.86, P=0.0302) (Fig. 3). However, the interaction between microbial and fertilizer application significantly influenced the PPO
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activity (F= 6.29, P=0.0006).
Regarding the tomato, the results indicated that microbial application (F = 4.82, P =
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0.0140) and the fertilization level (F= 26.74, P= 0.0001) significantly influenced the water content (Fig. 4). Moreover, the interaction between microbial and fertilizer application on the
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water content was not significant (F= 2.72, P=0.0446). Protein content did not differ
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significantly among the microbial treatments (F= 0.61, P=0.5515) (Fig. 4); however, the fertilization level significantly affected the protein content (F= 23.00, P=0.0001). The
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fertilization treatment contained a higher protein content than did the control treatment. However, the effect of the combined microbial and fertilizer treatment on the protein content
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was nonsignificant (F= 1.02, P=0.4093). The results revealed that PPO activity was not affected by microbial application (F= 4.64, P=0.0161) (Fig. 4), whereas the fertilization level significantly affected the PPO activity (F= 11.76, P=0.0001). Moreover, the effect of microbial and fertilizer application on PPO activity also did not differ significantly (F= 1.82, P=0.1463).
Insect Performance The results indicated that microbial and fertilizer application produced various effects 12
ACCEPTED MANUSCRIPT on the performance of S. litura larvae. Regarding the larvae that fed on the cabbage, the results indicated that microbial (F=4.03, P=0.0214) and fertilizer (F= 5.92, P=0.0040)
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application significantly affected the RGR (Fig. 5). Moreover, the interaction effect of
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microbial and fertilizer application on the performance of S. litura was significant (F= 4.31,
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P=0.0033).
Regarding the larvae that fed on the tomato foliage, the results indicated that the RGR
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was not significantly influenced by microbial treatment (F= 1.93, P=0.1511) (Fig. 5). However, the fertilization level significantly affected the performance of S. litura (F= 22.15,
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P=0.0001). The larvae grew 2 times faster on the foliage when using the full fertilizer treatment than when using the control treatment. In addition, the interaction between
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Discussion
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microbial and fertilizer application on RGR was significant (F= 3.08, P=0.0207).
The results of this study indicate that microbial application and fertilizer application
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produce significant and different effects on plant growth and insect performance. In addition, microbial treatment can promote plant growth and significantly reduce the amount of fertilizer applied.
Fertilizer may be the most crucial factor that affects plant growth and yield. Fertilizer application can promote plant growth. A previous study indicated that fertilized plants contain leaf numbers and plant biomass 5 times higher than those of unfertilized plants (Hsu et al., 2009). However, high level of fertilization may also lead to high insect damage levels. In addition to fertilizer, PGPMs in the soil environment have also been identified to play vital roles in plant growth. Studies have revealed that several species of PGPMs, such as Bacillus 13
ACCEPTED MANUSCRIPT sp. and Burkholderia vietnamiensis els., can significantly increase plant growth performance in numerous plant species (Kokalis-Burelle et al., 2002; Rekha et al., 2007). Fertilizer and
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PGPMs may independently facilitate plant growth and a combined effect may occur when
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they are used together. A previous study determined that combined PGPM and low fertilizer
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application produces plant biomass and nutrient uptake levels similar to those of full fertilizer treatment (Afzal and Bano, 2008; Adesemoye and Kloepper, 2009; Nakayan et al., 2013). Our results also indicated that microbials and fertilizer significantly influence plant growth.
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Moreover, we observed that plants treated with the fungi Meyerozyma guilliermondii and a
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half level of fertilization generate a plant biomass similar to that generated when using full fertilizer treatment, suggesting that adding these microbials can reduce the amount of
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fertilizer required and, consequently, reduce farming costs.
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Fertilizer and PGPM application may affect plants’ chemistry (Stout et al., 1998; Lucy et al., 2004; Hsu et al., 2009; Peric et al., 2009). Fertilizer normally exhibits a
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significant influence on the primary plant metabolites. Previous studies have indicated that high nutrient availability increases foliar protein and nutrient content (Stout et al., 1998;
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Prudic et al., 2005; Peric et al., 2009; Bybordi and Ebrahimian, 2013). The results of the current study also suggested that full fertilizer treatment produces twice the protein content. In addition, the levels of secondary plant metabolites, such as defensive proteins (trypsin inhibitor (TI) and PPO) and phenolics, can also be elevated by fertilization; this maybe due to an increase in resource availability (Stout et al., 1998; Hsu et al., 2009; Peric et al., 2009). The PPO activity of the tomato plant, however, decreased when applying fertilizer in this study, and the cause remains unclear. Moreover, because of their characteristic of enhancing root absorbing efficiency, PGPMs have been considered to influence the nutrient uptake of host plants and to increase the macro- and micronutrient and protein content of plants (Egamberdiyeva and Höflich, 2003; Glick, 2004; Bashan and de-Bashan, 2005; Lai et al., 14
ACCEPTED MANUSCRIPT 2008). PGPMs have been observed to increase the nitrogen content and mineral content of various plant species (Boddey and Dobereiner, 1988; Belimov and Dietz, 2000; Cakmakci et
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al., 2001; Dobbelaere et al., 2001; Probanza et al., 2002). Similarly, relevant literature has
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also indicated that PGPMs induce the production certain secondary plant metabolites
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(Figueiredo et al., 2011; Shanmugam and Kanoujia, 2011). Previous studies have determined that plants treated with PGPR increase the activity of peroxidase, PPO, phenylalanine ammonia-lyase, and proteinase inhibitors (Chen et al., 2000; Figueiredo et al., 2011;
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Shanmugam and Kanoujia, 2011). However, little is known about the interaction between the
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2 factors; only a few studies have indicated that the effect of PGPMs and fertilizer increases the concentrations of macronutrients (N, P, K, and Ca) and micronutrient (Mg, Fe, Zn, Cu,
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and Mn) in plants (Lai et al., 2008; Nakayan et al., 2013). Our results suggest that combined
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microbial and fertilizer treatment significantly increases foliar protein content. However, the PPO activity decreased during combined treatment. Therefore, the effect of PGPMs and
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fertilizer on the nutrient content of plants can be confirmed; however, the effects on plant defensive compounds remain unverified. Additional studies are required to clarify the effect
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and the mechanisms of PGPMs on the nutrient allocation of plants. Fertilizer and PGPMs are 2 vital factors that influence a plant’s chemistry and subsequently affect herbivorous insects’ performance (Ramamoorthy et al., 2001; Herm, 2002; Pineda et al., 2010). Previous studies have indicated that fertilized plants produce small amounts of defensive chemicals; thus, herbivorous insects perform better on fertilized plants than on unfertilized plants (Altieri and Nicolls, 2003; Prudic et al., 2005; Hsu et al., 2009). PGPMs promote plant growth by increasing root absorption; however, relevant literature has reported that PGPMs may also induce a plant’ defense responses (Ramamoorthy et al., 2001; Pineda et al., 2010). For example, a previous study revealed that PGPR-treated plants are resistant to feeding by the cucumber beetle Diabrotica undecimpunctata howardi Barber 15
ACCEPTED MANUSCRIPT (Zehnder et al., 1997; Zehnder et al., 2001), leaf folder Cnaphalocrocis medinalis (Saravanakumar et al., 2007), and leaf miner Aproaerema modicella (Senthilraja et al., 2010).
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Fertilization and PGPMs exhibit varied effects on plants and, consequently, on herbivorous
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insects; however, the combined PGPR and fertilizer effect on insect performance is largely
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unknown. Our results indicate that microbial application and fertilization could significantly affect the RGR of the third instar larvae of S. litura; but the interaction effect is still not clear. In summary, our study suggests that PGPMs and fertilizer exert various influences on
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plant growth performance, foliar chemistry, and insect performance. Both treatments exhibit
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positive effects on plant performance. However, the interaction effect of the microbials and fertilizer on plants remains unclear. Therefore, additional studies are required to clarify the
Acknowledgments
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interaction effect when using them together in promoting crop production and pest control.
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We thank Wallace Academic Editing and two anonymous reviewers for their
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comments on the manuscript.
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ACCEPTED MANUSCRIPT Table 1 Chemical properties of the field soil used in the study.
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EC (µS/cm)
30.9
OM (%)
0.34
Total N (g kg-1)
0.595
P (mg kg-1)
76.13404
K (mg kg-1)
35.75068
Ca (mg kg-1)
443.2
Mg (mg kg-1)
148.3595
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592.1538
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Cu (mg kg-1)
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Fe (mg kg-1) Mn (mg kg-1)
Zn (mg kg-1)
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pH
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Property Value ________________________________________________
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222.9418 6.513184 45.12512
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ACCEPTED MANUSCRIPT Table 2 The microorganisms used for the study.
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B1a
Bacteria (B)
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Rhizobium miluonense Rhizobium lusitanum
B2a
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Bacillus subtilis
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Burkholderia phytofirmans
Ochrobactrum pseudogrignonense
Fungi (F)
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B1 and B2, mixture of the bacterial strains.
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a
Meyerozyma guilliermondii
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Bacteria
Concentration
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_____________________________________________________________ Fa
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2nd inoculation
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B2b
3rd inoculation
F
B1
4th inoculation
F
B2
1:50
5th inoculation
F
B1
1:50
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1st inoculation
1:200 1:100 1:100
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F, fungi. b B1 and B2, different bacterial mixtures.
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a
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Fig. 1. Growth performance of cabbage plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test)
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Fig. 2. Growth performance of tomato plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test)
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Fig. 3. Chemical components of cabbage plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test) 30
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Fig. 4. Chemical components of tomato plants treated with microbials and fertilizer. Mean±SE (n = 5) (P < 0.05, Tukey’s test) 31
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B
Fig. 5. Relative growth rate of third instar larva of Spodoptera litura on cabbage plants (A) and tomato plants (B) treated with microbials and fertilizer. Mean±SE (n = 10) (P < 0.05, Tukey’s test)
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ACCEPTED MANUSCRIPT Highlights
We assessed the effect of PGPMs and fertilizer on plant and insect feeding.
Cabbage and tomato plants were inoculated with PGPMs (fungi and bacteria) and
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We found fungi Meyerozyma guilliermondii and fertilizer treatment promoted the
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various levels of fertilization.
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