Relation between plant nutrition, hormones, insecticide applications ...

Eur J Plant Pathol (2013) 137:727–742 DOI 10.1007/s10658-013-0283-7

ORIGINAL RESEARCH

Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing Weishou Shen & Juan M. Cevallos-Cevallos & Ulisses Nunes da Rocha & Hector A. Arevalo & Philip A. Stansly & Pamela D. Roberts & Ariena H. C. van Bruggen

Accepted: 19 August 2013 / Published online: 7 September 2013 # KNPV 2013

Abstract Intensive insecticide and nutrient management have been attempted worldwide to reduce citrus huanglongbing (HLB) symptom development and yield loss. However, effects of insecticide and nutrient applications on HLB have been poorly understood. Leaf nutrients, jasmonic and salicylic acid contents, Electronic supplementary material The online version of this article (doi:10.1007/s10658-013-0283-7) contains supplementary material, which is available to authorized users. W. Shen : J. M. Cevallos-Cevallos : U. Nunes da Rocha : A. H. C. van Bruggen (*) Emerging Pathogens Institute and Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA e-mail: [email protected] W. Shen Department of Environmental Science and Engineering, Nanjing Normal University, Nanjing 210023, China J. M. Cevallos-Cevallos Centro de Investigaciones Biotecnológicas del Ecuador (CIBE), Escuela Superior Politécnica del Litoral (ESPOL), Guayaquil 090112, Ecuador U. Nunes da Rocha Lawrence Berkeley National Laboratory, Earth Sciences Division, Berkeley, CA 94720, USA H. A. Arevalo : P. A. Stansly : P. D. Roberts Southwest Florida Research and Education Center, University of Florida, Immokalee, FL 34142, USA

cycle threshold (Ct) values of Ca. Liberibacter asiaticus (Las), and community structure of endophytic α-proteobacteria were evaluated after insecticide treatment, ‘nutrition’ treatment (including systemic resistance inducing agents), or both in comparison with a control in a two-factor field experiment in 2008–2012. Leaf N, Mn, Zn and B significantly increased whilst Cu decreased after nutrient applications. Salicylic acid significantly increased in old leaves treated with insecticides, nutrients or both, and in young leaves treated with nutrients only. The jasmonic acid concentration was highest after the nutrition treatment in both old and young leaves. Ct values of Las and leaf area and weight significantly increased after long-term nutrient applications in 2011 and/or 2012. Redundancy analysis of the endophytic α-proteobacteria community structure indicated that the communities were mainly separated according to nutrient applications, which were positively associated with Ct values of Las and Ca, Mn, Zn, B, Mg, and Fe contents in leaf samples collected in 2012. Thus, effects of insecticides on HLB were significant in the early 2-year period whilst nutrients had significant effects on Las content and leaf size and weight after at least 3 years of application. Keywords Boyd’s nutritional program . Ca. Liberibacter asiaticus . Cycle threshold (Ct) value . Induced systemic resistance (ISR) . Huanglongbing (HLB) . Systemic acquired resistance (SAR)

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Introduction Citrus greening disease or huanglongbing (HLB) is one of the most important citrus diseases worldwide (Bové 2006; Gottwald 2010), causing such severe losses that this disease is threatening commercial citrus production wherever the disease occurs (Gottwald 2010). This disease has long been known in Asia and Africa, but was found in South America only in 2004. In Florida, HLB was first detected in 2005 during a routine survey by regulatory agents, although the vector of the disease, the Asian citrus psyllid (Diaphorina citri Kuwayama, Sternorrhyncha: Psyllidae) was discovered as early as 1998 (Halbert and Manjunath 2004; Halbert 2005). Since then, the disease has spread through most of Florida, where the majority of the U.S. commercial citrus groves are located. In recent years, the vector and pathogen have spread to other South Eastern States in the U.S., and the pathogen was detected in California in 2012 (Stokstad 2012). HLB has not been found yet in Europe or the Middle East. Citrus greening is consistently associated with a bacterium that has not been cultured reliably yet. The putative causative agent is named Candidatus Liberibacter sp., belonging to the α-proteobacteria. Three species of Ca. Liberibacter are known to colonize citrus trees: Ca. Liberibacter asiaticus in Asian countries and since recently also in Eastern Africa and in the Americas, Ca. Liberibacter africanus in Southern Africa and some East African countries, and Ca. Liberibacter americanus in Brazil (Bové 2006; Gottwald et al. 2007; Mangomere et al. 2009; Teixeira et al. 2008). In the USA, the only species that has been found so far is Ca. Liberibacter asiaticus or Las (Gottwald et al. 2007). Ca. Liberibacter spp. are transmitted from tree to tree by psyllids, Diaphorina citri, in most citrus growing regions and Trioza eritreae in Africa. Both psyllid species can transmit all species of Ca. Liberibacter (Bové 2006). Adult psyllids are attracted to volatiles emitted from flushing shoots (Patt and Sétamou 2010), females lay eggs on young twigs, and nymphs develop on young twigs and leaves (flush). Ca. Liberibacter cells are taken up from the phloem through the mouthparts of nymphs and adults (Inoue et al. 2009). The bacteria can cross the gut membranes and move to the salivary glands from which they are injected into the phloem (Bonani et al. 2010). The psyllids remain infectious for life and can transmit the pathogen to their offspring (Pelz-Stelinski et al. 2010).

Eur J Plant Pathol (2013) 137:727–742

In Florida, the psyllid populations are highest in spring, but they can occur at any time of the year when trees produce new flush (Rogers and Ebert 2009). Management of D. citri populations and consequently HLB is currently based on intensive use of insecticides (commonly every 2 weeks). Even in winter, application of insecticides is recommended to minimize psyllid infestations on flush later on (Qureshi and Stansly 2010). An intensive areawide insecticide program is now in place in Florida, but HLB continues to be a major problem. Many different insecticides are available for managing the Asian citrus psyllid, and insecticides are rotated to limit the development of resistance in the psyllids. Nevertheless, insecticide resistance, in particular to imidacloprid, has already developed in field populations of D. citri in Florida (Tiwari et al. 2011), and negative side-effects on the parasitoid Tamarixia radiata, introduced into Florida for biological control of the psyllid, has also been observed (Hall and Nguyen 2010). Moreover, secondary pest outbreaks of various citrus scales and mealy bugs can be a real threat resulting from intensive insecticide use for psyllid control (Wakgari and Giliomee 2003). In addition, the insecticides commonly used for the control of the Asian citrus psyllid have not been as effective as expected (Ichinose et al. 2010). Finally, insecticides may be helpful in reducing the spread of Ca. Liberibacter asiaticus in the beginning of the epidemic, but may not reduce the pathogen load in the trees once most trees in a grove are infected (Chiyaka et al. 2012). After transmission of Ca. Liberibacter asiaticus to citrus, infected trees respond to the bacteria with a wide array of physiological reactions resulting in a sequence of symptoms, from mild chlorosis to distinct mottling, associated with an increase in bacterial concentrations (Coletta-Filho et al. 2010). A hormonal imbalance may be responsible for the development of lopsided fruits with uneven colouring (Kim et al. 2009). Starch is accumulated in leaves and stems as the phloem gets blocked (Etxeberria et al. 2009; Kim et al. 2009). As a consequence, roots starve and can become infected by secondary pathogens and mineral nutrition is out of balance (Pereira and Pereira Milori 2009). Within 2– 3 years after inoculation a tree may die. Despite this general trend in disease development, variation in the extent of plant response to infection can be found. This can be due to differences in genotype (Folimonova et al. 2009), but some trees with the same genotype can also differ in disease reaction, suggesting that some other factors may be responsible for relative resistance


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to HLB. One factor that may influence the resistance to disease can be the nutritional condition, which can influence the microbial community in the rhizosphere and endosphere of plants (Dordas 2008; Gu et al. 2013). In turn, bacterial and fungal endophytes can change the phloem composition and induce systemic resistance (Musetti et al. 2007). A reduction in HLB symptom development and yield loss has been attempted by intensive nutrient management. In particular, micronutrients combined with salicylic acid and/or phosphite have been applied, often as foliar sprays, to maintain the productive capacity of HLB infected trees (Ahmad et al. 2011; Masaoka et al. 2011; Razi et al. 2011). The effectiveness of these treatments has been controversial (Gottwald et al. 2012), but balanced nutrition likely slows down tree decline due to HLB, considering the occurrence of healthy looking trees many years after Las infection was demonstrated (field observations by the authors). Asymptomatic infection has been noticed in several organically managed groves, despite infestation by Las-positive psyllids (Halbert, unpublished; Shen et al. 2013). The nutritional programs employed vary tremendously and mostly have not been tested in replicated experiments (including controls) over many years. One of these programs (Table 1), developed by citrus grower Maury Boyd, has been instituted since the start of the epidemic in 2005. Trees treated according to the Boyd program look healthier than untreated trees and have adequate fruit production, despite being 100 %

infected with Las. The Boyd program has been tested in a replicated experiment in combination with insecticide treatments and a negative control since 2008 (Stansly et al. 2011). In the first 3 years no effects were detected of the nutrient plus salicylic acid treatments on the Las content in the trees or on yield (Stansly et al. 2011). In another replicated (but shorter-term) experiment no differences were observed in tree health, fruit quality and production, or Las titre between trees treated with a nutritional program (plus or minus phosphite) and a control treatment (Gottwald et al. 2012). Additionally, application of micro nutrients, phosphite and salicylic acid was compared to regular nutrient management in several commercial blocks, but no differences in disease development or yield were observed. Both of these experiments lasted 2 years (Gottwald et al. 2012). Thus, it has not been shown so far if nutritional programs can lower the Las content and reverse its effects on citrus trees in the long run. The Boyd nutritional program includes agents that induce systemic resistance, either systemic acquired resistance (SAR) or induced systemic resistance (ISR), but individual effects of these agents on HLB have not been published in scientific journals. Nevertheless, soil-applied imidacloprid, isonicotinic acid and acibenzolar-s-methyl did elicit SAR in citrus trees in greenhouse tests, and reduced the severity of citrus canker (Francis et al. 2009). Boyd’s nutritional program is commonly combined with the application of insecticides. The individual

Table 1 Composition of the foliar spray known as ‘Boyd nutrient solution’ Product

Unit per acre

Impact on microbes or plants

Reference

13-0-44 fertilizer

8.5 lb

N-K fertilizer

Diamond R

Techmangan (Mn sulfate)

8.5 lb

Mn fertilizer

Diamond R

Zinc sulfate

2.8 lb

Zn fertilizer

Diamond R

Epsom salts

8.5 lb

Mg fertilizer

Diamond R

Sodium molybdate

0.85 oz

Mo fertilizer, bactericide

Diamond R

Di-oxy solv organic (hydrogen dioxide)

2 qt

Oxidizer (fungicide/bactericide)

Flo-Tec Inc.

3-18-20 with K-phite (potassium phosphate)

8 gal

Plant Food Systems

Serenade Max WP (Bacillus subtilis)

2.25 lb

AgraQuest, Inc.

PetroCanada

SAver (salicylic acid)

1 qt

Oomycte inhibitor, induced systemic resistance (ISR) Biological fungicide/bactericide, Systemic acquired resistance (SAR) inducer SAR inducer

435 oil

5 gal

carrier

Plant Food Systems

Citrus grower Maury Boyd developed a program with multiple applications of various nutrients (as listed above plus Borate and Canitrate), hydrogen dioxide, potassium phosphite, Bacillus subtillis, ammonium and potassium salicylate, and 435 spray oil

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effects of insecticide treatments and a nutritional program have not been studied in detail. In 2008, Stansly and colleagues started a long-term replicated field experiment testing effects of insecticides, Boyd’s nutrient program and both of these in comparison with a non-treated control treatment. HLB had just been detected in some trees in the experimental grove at that time. In the first few years (2008–2010), effects of insecticide treatments on HLB development and yield were significant, but there were no effects of the nutrient treatment (Stansly et al. 2013). The current study was carried out with the goal to investigate if prolonged treatment with nutrients (including ISR and SAR inducing agents) would have an effect on endophytic bacterial communities, Las titres, plant hormone concentrations and leaf sizes. Our hypotheses were: (1) in the long run, insecticides have a different effect on microbial communities than ‘Boyd nutrient solution’ compared to the control treatment; (2) insecticides do not affect Las titres once most of the trees are infected; (3) ‘Boyd nutrient solution’ enhances the diversity of endophytic bacteria, induced systemic resistance and/or systemic acquired resistance, and foliar growth; (4) a combination of both insecticides and ‘Boyd nutrient solution’ is the best agricultural practice in areas where a limited number of the trees are infected with Las. To test these hypotheses, soil and leaf samples were collected from the experimental grove mentioned above (Stansly et al. 2013), and were analyzed for nutrients, endophytic microbial communities, Las titers, plant hormones and leaf sizes.


two main components: foliar nutrient solution (N1) and untreated (N0), and insecticide (I1) and untreated (I0). The combination of these two main factors determined the four treatments: control (N0I0), nutritional of Moury Boyd or MB (N1I0), insecticide or INS (N0I1), and MB + INS (N1I1). Insecticide applications

Material and methods

Chemical applications were timed by Integrated Pest Management (IPM) guidelines, and applied on an “as needed” basis according to observations made during weekly scouting. Five foliar insecticide applications were administered during each growing season in the insecticide and ‘nutritionals plus insecticide’ plots: (1) Danitol® 4 E.C. (fenpropathrin) [Valent USA Corp. Walnut Creek, CA] at 0.9–1.2 l/ha, (2) Delegate™ WG (spinetoram) [Dow Agrosciences. Indianapolis, IN] at 0.3–0.4 l/ha, (3) Mustang ® (zeta-cypermethrin) [FMC. Philadelphia, PA] at 0.3 l/ha, (4) Movento® (spirotetramat) [Bayer CropSciences. Research Triangle Park, NC] at 0.7–1.2 l/ha, and (5) Lorsban 4E (chlorpyrifos) [Dow Agrosciences. Indianapolis, IN] at 3.5 l/ha. The products were selected based on the time of the year and current management guidelines for Asian citrus psyllids. All of the insecticide treatments included 2 % of 435 horticultural oil. All applications were made using an air blast sprayer at 10 bars, averaging 982 l/ha. In addition, Temik 15G (aldicarb) [Bayer CropSciences, Research Triangle Park, NC] was applied at 22–34 kg/ha each winter according to the guidelines followed by commercial growers.

Experimental field and design

Nutrient applications

The experiment was conducted in block B9 in Silver Strand’s North Grove, a 5.2 ha block of ‘Valencia’ orange on ‘Swingle’ citromelo rootstock planted in June 2001. The site is located in Collier Co. (26° 29′N 81° 21′W, Florida, USA). The soil type is primarily Immokalee fine sand, with a slightly different soil type, Basinger fine sand, mostly confined to block 2 (Fig. 1). The annual precipitation is 117–137 cm, and the average annual air temperature is 21–25 °C. In February 2008, 2 years after the detection of HLB, the block was divided into 16 plots to fit a randomized complete block design with four replicates and four treatments (Fig. 1). Treatment selection corresponded to a 2×2 factorial experiment using the

Fertilizers (NPK or as listed) were applied to soil as follows: 13-0-21 at 336 kg/ha in September 2008; 124-16 at 448 kg/ha in January 2009; 8-0-24 at 448 kg/ha in May 2009; K-Mag® (22 % K2O, 11 % Mg and 22 % S) at 224 kg/ha in October 2009 and August 2010; UN-32 (45 % NH4NO3, 35 % urea and 20 % water) at 186 l/ha in October 2009, January and April 2010; 0-0-42 at 224 kg/ha on March 2010, May and August 2011; 9-0-0 liquid at 93 l/ha in March 2010; Granulite (heat dried biosolids) at 1,120 kg/ha in May 2010; 14-0-22 at 336 kg/ha in September 2010; 16-416 at 336 kg/ha in January 2011; 20-0-0 plus 5 % Ca liquid at 96 l/ha in May and August 2011.


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MB = Nutritional Ctrl = Control Ins = Insecticide Ins+MB = Combined insecticide + Nutritional Bk: 1 Trt: MB

Bk: 1 Trt: Ins + MB

Bk: 2 Trt: MB Bk: 2 Trt: Ctrl

Bk: 1 Trt: Ctrl

Bk: 1 Trt: Ins

Bk: 2 Trt: Ins + MB

Bk: 3 Trt: Ins

Bk: 2 Trt: Ins Bk: 3 Trt: Ctrl Bk: 3 Trt: Ins + MB

Bk: 4 Trt: MB

Bk: 3 Trt: MB

Bk: 4 Trt: Ctrl Bk: 4 Trt: Ins + MB Bk: 4 Trt: Ins

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Fig. 1 Field layout of an experiment testing the effects of insecticide applications, nutritional management (Maury Boyd, MB) or both on ‘Valencia’ orange on ‘Swingle’ citromelo

rootstock compared to non-treated controls. The experiment was conducted in Silver Strand’s North Grove just North of Immokalee in South Florida from 2008 to 2012

‘Boyd nutrient solution’ (Table 1) was sprayed on the foliage in designated plots (nutrition only and insecticide plus nutrition treatments) three times a year when major flushes were fully expanded but not hardened. Applications were performed with an Air-O-Fan airblast sprayer equipped with Albuz® ATR hollow cone nozzles providing an 80o spray pattern with five blue and one green nozzle (2.5 and 3.4 l/min,

respectively) at 10 bars and 5.2 km/h, delivering a total 39 l/min or 982 l/ha (105 gal/ac). Sampling In 2010, soil samples were collected under one randomly selected tree from each of the 16 plots. Each sample consisted of four subsamples around the

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selected tree pooled into plastic bags. The soil was transported from the field to the laboratory in Gainesville in an ice cooled container. All leaf samples were collected from trees of the same size and age; young, replanted trees were avoided. In 2011, pooled leaf samples were collected from one tree per plot and transported in liquid nitrogen to the laboratory in Gainesville for plant hormone analysis. In October 2011, leaf samples were collected from six to 17 random trees in each of the 16 plots, totaling 148 samples. The samples were placed in a cool box with ice and brought to the HLB laboratory at the SWFREC for DNA extraction and real-time qPCR analysis. In May 2012, leaf samples were collected from four random trees in each of the 16 plots, and transported in dry ice to the laboratory in Gainesville for analysis of plant nutrients, HLB titre by real-time qPCR, and microbial communities by PCR-DGGE (denaturing gradient gel electrophoresis). Soil and plant nutrient analyses Air-dried soil samples were subjected to analysis in the Soil Analysis lab at the University of Florida (UF), Gainesville, FL. Soil pH was determined with a glass electrode (soil:water = 1:2). Soil organic C was determined by the dichromate oxidation and total N by the Kjeldahl digestion according to Mylavarapu and Moon (2007). Automatic colorimetric analysis was employed to determine NH3-N and NOx-N (NOx-N = NO2-N + NO 3 -N) using an Alpkem Flow Solution IV autoanalyzer (OI Analytical, College Station, TX, USA). Soil P, K, Ca and Mg were extracted with Mehlich-1 extraction solution (soil:water = 1:4), and filtered through Whatman 42 filterpaper as described by Mylavarapu and Moon (2007). The filtrates were analyzed for nutrients using an inductively coupled plasma (ICP) spectrophotometer (SPECTRO Analytical Instruments Inc., Mahwah, NJ, USA). Composite leaf samples consisting of 20 leaves from each of 4 trees per treatment and per block were ground in a Foss Cyclotec 1093 (Eden Prairie, MN 55344), and then subjected to nutrient analysis in the Soil Analysis lab at UF, Gainesville. Nitrogen was analyzed using the total Kjeldahl nitrogen (TKN) method according to Mylavarapu and Moon (2007). Automatic colorimetric analysis was employed to determine nitrogen in TKN digestates using an Alpkem Flow Solution IV autoanalyzer (OI Analytical, College


Station, TX, USA). Other nutrients were analyzed using an ashing and acid digestion procedure as described by Mylavarapu and Moon (2007). The filtrate was analyzed for nutrients using an ICP spectrophotometer (SPECTRO Analytical Instruments Inc., Mahwah, NJ, USA). Plant hormone analysis Hormone analysis was exactly as in Birkemeyer et al. 2003. Briefly, 300 mg of frozen leaves were ground under liquid nitrogen and suspended into 3 ml Bieleski solvent pre-cooled to −20 °C and incubated at room temperature for 1 h. After centrifugation (5,000 × g for 5 min), the supernatant was separated and transferred to a vacuum centrifuge at 45 °C and 10 mbar until dryness. The dried residue was suspended in 30 μl of methanol and then, 200 μl diethyl ether were added and sonicated in a bath for 1 min. Samples were then applied to an aminopropyl solid-phase extractioncartridge (Fisher Scientific, St Louis, MN, USA). Each column was preconditioned with two times the volume of methanol/diethyl ether followed by sample application into each column. Columns were then washed with 250 μl of CHCl3:2-propanol = 2:1 (v/v), and the hormone fraction was eluted twice with 200 μl diethyl ether containing 2 % acetic acid. The combined eluted fractions were mixed with 3 μl of a 0.1-mg/ml solution of the internal standard 5α-cholestane and then taken to dryness in a vacuum centrifuge (1 min at 200 mbar, then for a further min at 10 mbar). The dried samples were suspended in 80 μl of N-methyl-N-(tert.butyldimethylsilyl) trifluoroacetamide (MTBSTFA) and incubated at 100 °C for 1 h. GC-MS analyses were carried out in a HP5890 GC coupled to an HP5971 series mass spectrometer (MS) from Hewlett Packard, (Santa Clara, CA). Chromatogram analysis was completed using HP ChemStation software. Samples (1 μl) were injected splitlessly at 230 °C with an oven, temperature ramp of 6 °C/min from 70 to 350 °C, the ion source temperature was set to 230 °C, and the transfer line was at 260 °C. Hydrogen carrier gas was used at a flow-rate of 1 ml/min. Quadrupole GC-MS chromatograms were by electron impact ionisation and total ion monitoring, m/z 40–600, as well as selective ion monitoring was used with the selected fragments of m/z 133 (JA), m/z 309 (SA), and m/z 217 (5α-cholestane). All reagents were from Fisher Scientific (St Louis, MN, USA).


Nucleic acid extractions For HLB testing, midribs of leaves and petioles were cut with a flamed scissor into 1–2 cm long pieces, pooled per sample, and placed in polypropylene vials prior storing at −80 °C. For microbial community analysis of the endophytes inside plant tissues, leaf samples were washed with sterilized-distilled water for 5 min using an ultrasonic cleaning system. The leaf samples were surface-sterilized for 30 s with 75 % ethanol, 5 min in a 5 % NaClO, followed by several washes with sterilized-distilled water. The leaf blades were cut into small pieces (about 2 cm diameter) and stored at −80 °C before DNA extraction. DNA from leaf samples was extracted with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Briefly, 100 mg of leaf midribs and petioles were added into XXTuff Reinforced Microvials (Biospec, Bartlesville, OK, USA) containing four 2.3 mm Chrome-Steel Beads (Biospec, Bartlesville, OK, USA). After the addition of liquid nitrogen, the samples were homogenized at 3,000 rpm for 1 min using a Powerlyzer 24 Bench Top Bead-Based Homogenizer (MO BIO Laboratories, Carlsbad, CA, USA). The sample materials were lysed, salt-precipitated, and centrifuged through the DNeasy Plant Mini Kit columns. Total DNA was purified with a silica-based membrane and elution reagent (QIAGEN Inc., Valencia, CA, USA). The purified DNA was stored at −20 °C for further analysis. Regular PCR and real-time PCR of Las Regular PCR was performed using primer pair OI1 and OI2c to detect Las in leaf samples (Jagoueix et al. 1996). Real-time PCR of leaf DNA was carried out using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) according to Li et al. (2006). The primer pair HLBas and HLBr, and a TaqMan probe HLBp were used for PCR amplification to target the 16S rRNA gene of Las (Li et al. 2006). An additional primer-probe set designed on the basis of the COX gene was used as positive internal control (Li et al. 2006). Two-step thermal profiles consisted of 95 °C for 20 s, followed by 40 cycles of 1 s at 95 °C and 40 s at 58 °C, with plate reading at 58 °C for data acquisition. Each run contained one positive and one negative control sample from citrus plants in a quarantine greenhouse

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at the Division of Plant Industry at Gainesville, FL (obtained from Debra Jones). Data analysis was performed with CFX Manager Software Version 2.0 (Bio-Rad Laboratories, Hercules, CA, USA). The HLB laboratory at the SWFREC considers positive to HLB samples with Ct values less than or equal to 32. PCR-DGGE analysis A nested system was used for amplification of αproteobacterial 16S ribosomal RNA genes. The first round of PCR was performed by applying the primer pair F203α (CCG CAT ACG CCC TAC GGG GGA AAG ATT TAT) and R1494 (CTA CGG YTA CCT TGT TAC GAC) (Weisburg et al. 1991; Gomes et al. 2001). The first round of PCR products then served as templates for a second round of PCR with primer pair F984GC (CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G AAC GCG AAG AAC CTT AC) and R1378 (CGG TGT GTA CAA GGC CCG GGA ACG) (Heuer et al. 1997), which amplified the variable V6 region of 16S rRNA. PCR was carried out with a Veriti 96 well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in 50 μl reaction volumes containing 0.4 μM of each primer, 2.5 mM MgCl2, 0.2 mM of each dNTP and 1.25 U Taq DNA polymerase (Invitrogen, Grand Island, NY, USA). DGGE was performed at 60 °C with the DCodeTM Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. DGGE was carried out using 6.5 % (wt/vol) polyacrylamide gels (ratio of acrylamide to bisacrylamide, 37.5:1) with a gradient of 40 % to 55 % where a 100 % denaturing solution is defined as 7 M urea and 40 % formamide (Watanabe et al. 2001). The gels were electrophoresed at 65 V for 16 h in 0.5 × TAE buffer and stained with SYBR Gold (Molecular Probes, Eugene, Oregon, USA) for 30 min (Tuma et al. 1999; Sigler et al. 2004). The stained gels were immediately photographed on a UV transilluminator with a CCD camera (Bio-Rad Laboratories, Hercules, CA, USA). Digital images of the gels were further analysed by Quantity One® 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, USA). Removing the background intensity from each lane, the software performs a density profile through lanes, detects individual bands and matches bands occupying the same position in different lanes (Xue

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et al. 2006). The genetic diversity of endophytic microbial communities was analyzed by Richness (S), Shannon diversity (H), and Evenness (EH) indices (Xue et al. 2006). Statistical analyses Statistical analyses were performed to test main effects, their interaction, and individual treatments using the General Linear Model Procedure. Main effects were considered if the interaction of the two factors was not significant (p>0.05). Results were subjected to analysis of variance (ANOVA) to determine the significance of the differences in data (plant hormones were Log10 transformed) with SAS 9.2 Software (SAS Institute Inc., Cary, NC, USA). Least significant difference (LSD) post-hoc tests were conducted to determine the differences between the individual treatments. Correlations of microbial communities of endophytic α-proteobacteria to environmental factors were assessed using redundancy analysis (RDA). RDA was performed using the ‘species data’ (individual DGGE bands) and ‘environmental data’ (leaf nutrient contents, leaf area, leaf fresh weight per unit of area, and leaf Las titer) with the computer software CANOCO 4.5 (Microcomputer Power, Ithaca, NY, USA). A Monte Carlo permutation test was carried out based on 499 random permutations.

Results Soil pH, organic C and nutrient contents Analyses of soil pH (p=0.001), NOx-N (p=0.002), and K (p=0.008) revealed significant interactions of the two factors (insecticides and nutrients), so that individual treatment combinations are reported separately. Soil pH was highest in control plots, lower in insecticide treated plots and nutrition plus insecticide plots, and lowest (p=0.05) after the treatment with Boyd’s nutrients (Table 2). Soil organic C was significantly decreased after the insecticide applications (p