Preliminary research on soil conditioner mediated citrusHuanglongbing mitigation in the field in Guangdong, China

Meirong Xu & Meidan Liang & Jianchi Chen & Yulu Xia &

Zheng Zheng & Qing Zhu & Xiaoling Deng

Accepted: 7 June 2013# KNPV 2013

Abstract Huanglongbing (HLB, yellow shoot disease)is devastating citrus production worldwide. No effectivecontrol measure is currently available. In China, man-agement of HLB through nutrient applications was usedin the past and the interest has been renewed recently. Inthis study the effects of a soil conditioner (SC) on HLB-affected mandarin citrus (Citrus reticulate Blanco) wereevaluated in four orchards (three with cultivar Shatangjuand one with cultivar Chuntianju) in GuangdongProvince, China. We observed that HLB trees showedmore vigorous growth with lower titres of “CandidatusLiberibacter asiaticus”, the putative pathogen of HLB,than the untreated trees at 10 months post treatment(MPT) with SC. “Ca. L. asiaticus” titres in younger

trees (2-year) were significantly lower than those inolder trees (8-years old) (P=0.005). Comparison of SCtreatments using 4-year-old Chuntianju showed that the“Ca. L. asiaticus” titres were significantly lower in newleaves with 7 MPT (94.51 % reduction or 2.59 timeslower) than those with 1 or 4 MPT (P=0.002). The P, N,K, Mn, and organic matters contents in the treatedorchard soils were all significantly higher than those inthe non-treated soil tested at 2 MPT (p<0.05). Effects ofSC treatments on fruit yield and quality were also sig-nificant. Our preliminary results showed that SC treat-ment could have various levels of positive influence onHLB-affected mandarin trees.

Keywords Huanglongbing . Citrus greening . Yellowshoot disease . Soil conditioner (SC) . “CandidatusLiberibacter asiaticus”

Introduction

Citrus is one of the most important fruit crops widelygrown in tropical and subtropical regions of the world,with a total global production reported to be over 120million tons currently. China is an important citrus-producing country and its citrus industry has beensteadily progressed for decades. However, as in mostcitrus production regions in the world, citrus produc-tion in China is threatened by Huanglongbing (HLB,yellow shoot disease), a devastating disease first ob-served in Guangdong Province over a hundred years

Eur J Plant PatholDOI 10.1007/s10658-013-0238-z

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10658-013-0238-z) contains supplementarymaterial, which is available to authorized users.

M. Xu :M. Liang : Z. Zheng :Q. Zhu :X. Deng (*)College of Natural Resources and Environment,South China Agricultural University,Guangzhou City, Guangdong 510642, Chinae-mail: xldeng@scau.edu.cn

Y. XiaThe Center of Integrated Pest Management,North Carolina State University,Raleigh, NC, USA

J. ChenSan Joaquin Valley Agricultural Sciences Center,United States Department of Agriculture,Agricultural Research Service,Parlier, CA 93648, USA

ago (Lin 1956). HLB is associated with “CandidatusLiberibacter asiaticus”, a nutritionally fastidious bacte-rium transmitted by the Asian citrus psyllid,Diaphorinacitri Kuwayama (Bové 2006).

HLB has no known effective cure. The lack of HLBresistance in major citrus species and cultivars has alsobeen a major impediment to breeding HLB-resistantgermplasm and varieties. Removal of diseased treeshas been recommended but may not be practical fortrees still in production. In Guangdong, managementof HLB-affected trees depends on the disease severityand tree age. Due to the impact of HLB, there are feworchards with citrus trees older than 15 years inGuangdong (Deng et al. 2012). In fact, most farmershave limited knowledge about HLB and its vector.Nutrition deficiency or other abiotic stressed trees areoften confused with HLB-affected trees. Therefore,both diseased and healthy trees were managed at thesame level. Therefore, increased cultivation measures,including the use of fertilizers/nutrients to compensatethe negative impact of HLB on trees, are generallyemployed.

Roles of nutrients in plant disease developmenthave been studied for years (Huber and Graham1999; Barker 2009). Huber and Haneklaus (2007)discussed the interactions of plant, pathogen, and en-vironment and noted that nutrition influenced allinteracting components and determined disease sever-ity. Nitrogen (N) contents in the soil organic matterand in the mature trees played an important role in theregulation of N supply to the growing parts of thecitrus tree (Dasberg 1987; Martínez-Alcántara et al.2012). Appropriate levels of soil P and K were criticalfor citrus production (Mattos et al. 2006). Citrus treesgrowing in soil containing higher levels of mineralshad greater concentrations of Zn and Fe in their leavesand lower levels of disease (Zhang et al. 2007). InChina, nutritional approaches, in conjunction withother cultural practices, can maintain grove productiv-ity for a certain time depending on the type of citrusspecies/cultivars, the age of the trees, the propagationmethod of the plants, the Asian citrus psyllid population,and possibly other factors (Xia et al. 2011). In addition,better agricultural traits like total contents of sugar andascorbic acid, and fruit size were correlated with properapplication of Zn, B, and Mo (Ouyang 1990).

A common method for improvement of soil nutri-ents and tree productivity is the application of soilconditioners (SCs). SCs are synthetic or natural

products that improve the soil’s physical qualities,especially the ability to provide nutrition for plants(Seybold 1994). SCs are often supplemented withnutrients. In a broad sense, a wide variety of materialssuch as compost, manure, and charcoal have beenused as SCs for their capacity to improve soil proper-ties and to supplement nutrients. Charcoal applicationsto soil were reported to promote citrus growth andtrigger vesicular-arbuscular mycorrhizal (VAM) de-velopment (Ishii and Kadoya 1994). There has notbeen a systematic study on how a SC influences HLBtrees.

The aim of this study was to evaluate the effects of aSC in reduction of HLB symptoms and “Ca. L. asiaticus”titres and increase of yield and fruit quality under fieldconditions in Guangdong, China. Meanwhile, changes ofnutrients in treated soil and expression of genes reportedto be involved in HLB development were also monitoredto search for molecular mechanisms of SC in controllingHLB.

Materials and methods

Plant materials

HLB-infected citrus trees with systemic distributionfrom five different orchards in the cities of Sihui,Qingyuan and Longmen, Guangdong Province, China,were selected for three experiments: 1) Two-, 3-, and 8-year old Shatangju (Citrus reticulate Blanco cv.Shatangju) trees in three different orchards of SihuiCity were used to study “Ca. L. asiaticus” titre varia-tions among trees of different ages; 2) Four year-oldChuntianju (Citrus reticulate Blanco cv. Chuntianju)trees in an orchard in Longmen City were used to study“Ca. L. asiaticus” titre variations within a growing sea-son; and 3) Six-year old Shatangju trees in an orchard inQingyuan City were used for evaluating fruit yield andquality along with a soil nutrition analysis. HLB affect-ed trees were identified by symptomatology and con-firmed by nested PCR detection of “Ca. L. asiaticus”(Deng et al. 2007), as well as real-time PCR (Li et al.2006) detection of “Ca. L. asiaticus”. A randomizedblock experiment was designed in each orchard. Six or10 plants were selected from each orchard. Half of eachgroup of selected trees was treated with the SC, the otherhalf were untreated controls. The experimental parame-ters are listed in Table S1.

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Soil conditioner

The SC used in this study was provided by ZaoqingSouth China Run-zhe-xin-ye Biotechnology LimitedLiability Company. The SC had the following proper-ties: pH=4.3, moisture=7.76 %, nitrogen (N)=0.63 %,phosphorus (P)=0.30 %, potassium (K)=0.07 %, andorganic matter=10.39 %. SC powders were broadcastedaround the range of the feeder root crown, mixed intosoil (about 15 cm depth) by farm tools, and flooded with5 to 10 l of water immediately.

Sampling methods

Three scaffold brancheswere selected on each tree. Youngleaves at top and mature leaves at the base in each branchwere collected. For evaluating bacterial titre as relatedto tree age (Experiment 1), sampling was performed2 months post treatment (MPT). For evaluating the rela-tionship of bacterial titre and the growing season(Experiment 2), samplings were performed at 1, 4, 7,and 10 MPT. For determining the effects of soil charac-teristics on yield (Experiment 3), all fruits were harvested10MPT for yield recording and nutrient test. Soil sampleswere collected at 2 MPT for microbial test (Table S1).

Quantification of “Ca. L. asiaticus” by real-time PCR

For DNA extraction, midribs of samples were cut intosmall pieces at room temperature. Exactly 0.1 g oftissue was ground into fine powder with a sterilizedmortar and pestle in liquid nitrogen. DNA wasextracted using DNeasy Plant Mini Kit (Qiagen,Valencia, CA) according to the manufacturer’s instruc-tions, quantified with the Agilent Model 2100Bioanalyzer (Agilent Technologies, Palo Alto, CA),adjusted to a final concentration of 35 ng μl−1 for eachsample, and stored at −20 °C for further use.

Quantitative real time PCR (qRT-PCR) wasperformed using TOYOBO Realtime PCR MasterMix (QPK-101) according to the manufacturers’ in-structions. The primers and probe used in this studywere described earlier (Li et al. 2006). Amplificationwas carried out in a 20 μl reaction mixture containingapproximately 35 ng of DNA template, 2× pre-mix,and 10 μM of each forward and reverse primers andthe TaqMan probe. The qRT-PCR was assessed in aThermal Cycler Dice Real Time System (TaKaRaCode: TP800, Osaka, Japan). All reactions were

performed in duplicate and each run contained onenegative (DNA of healthy citrus) and one positive con-trol, a recombinant plasmid clone with “Ca. L. asiaticus”16S rDNA generated by primer set OI1/OI2c (Jagoueixet al. 1994). A standard curvewas generated according to10-fold serial dilutions of the OI1/OI2c plasmid withconcentrations ranging from 8×100 to 8×106 fg μl−1.

Comparative gene expressions in SC- and non-SCtreated trees

Leaf samples were collected from the Shatangju man-darin trees on the 2 and 3 year old orchards in SihuiCity (Experiment 1). Total RNAwas extracted follow-ing SDS methods (Chattopadhyay et al. 1993; Zhanget al. 2003) with some modifications. Briefly, 0.5 g ofequal portions of mature and young leaves were groundin 2.7 ml of extraction buffer mixed with 2 % SDS,0.02 mol l−1 Borax, 4 % β-Mercaptoethanol, 3 % Tris-phenol, and 2.2 % Chloroform. Fine tissue was vortexedfor 3 min at 6,000 rpm, centrifuged at 12,000 rpm for5 min (4 °C). The aqueous phase was mixed with 0.6 mlof Tris-phenol and chloroform-vortexed and centrifugedrespectively. The RNAwas precipitated with 4 M LiCland ethanol, washed once with 75 % Ethanol, dried atroom temperature and dissolved in 60 μl RNase-freewater (TaKaRa, Code No.: D9012). All RNA sampleswas qualified by Agilent Model 2100 Bioanalyzer andkept in −70 °C for further use.

Target genes and their primers were selected based onprevious publications (Albrecht and Bowman 2008; Kimet al. 2009; Fan et al. 2011; Albrecht and Bowman 2012;Maul et al. 2007; Xiao et al. 2011) (Table 1). Three μgRNA of each sample was reverse-transcribed usingReverTra Ace® qPCR RT Kit (FSQ-101, Toyobo Co.,Ltd., Osaka, Japan). DNase I (D2270A, Takara Co. Ltd.,Osaka, Japan) was used to remove residual genomicDNA. The cDNA samples were adjusted to the concen-tration of 1,000 ng Μl−1. Conversional PCR wasperformed in a PTC-100 or PTC-200 Thermal Cycler(MJ Research) with an initial 5 min denaturation at95 °C, followed by 30 cycles of each (95 °C, 30 s;55 °C, 30 s; 72 °C, 30 s), then extended at 72 °C for7 min. Amplicons were visualized after 1.2 % agarosegel electrophoresis and staining by 5 % goldview(BioRoYee Co., Ltd., 15–3003). Each sample was doneby triplicate. Up- or down- regulation of a gene in a SCtreatment was determined by comparison of ampliconfluorescence intensity to that of the non-SC treatment.

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Soil microbial counts and nutrient analyses

Soil samples from the orchard in Qingyuan City(Experiment 3) were collected 2 MPT. Ten g of eachsample were prepared for plating by blending in 20 mlsterilized water. Serial dilutions of soil suspensionwere then prepared and the 10−3 and 10−4 dilutionswere spread-plated in triplicate on each of the threemedia: gauze No. 1 (GA) for actinomycetes, Nutrient

agar (NA) for other bacteria, and potato dextrose agar(PDA) for fungi. The actinomycete, bacteria, and fun-gi colonies were counted after incubation at 26 °C forbacterial and 28 °C for fungi. The total number ofcolonies was converted to number of colonies per gof soil. The diversity of colonies was evaluated bycolony morphology.

For nutrient analyses, equal amount of soil sampleswere collected from five sites (east, west, north, south,

Table 1 Expression profiles of 25 selected citrus genes in HLB affected 2 and 3 year-old citrus trees treated and non-treated with soilconditioner (SC) at 2 months post treatment (MPT)

Genome locus Description 2 year treea 3 year treeb Reference

SC Non-SC SC Non-SC

Up-regulated related to SC treatment:

Cit.7043.1.S1 Miraculin-like protein 1 gene Fan et al., 2011

Cit.2831.1.S1 The plant defensin family (PDF2.2) Albrecht and Bowman, 2012

CX045772 Putative cell death associated protein Kim et al., 2009

Cit.8866.1.S1 Vitamin C defective 2 (VTC2) Albrecht and Bowman, 2012

Cit.165.1.S1 Chlorophyll A-B binding family protein Albrecht and Bowman, 2008

Cit.31971.1.S1 Myb-like HTH transcriptional regulator protein Albrecht and Bowman, 2012

Cit.28102.1.S1 Chloroplastic copper/zinc superoxide dismutase Albrecht and Bowman, 2012

CF653559 Pathogenesis-related protein PR-1 precursor Kim et al., 2009

Down-regulated related to SC treatment:

Cit.28102.1.S1 Cu/Zn superoxide dismutase gene Fan et al., 2011

DT214451 Beta-amylase Kim et al., 2009

CX639454 Plant glycogenin-like starch initiation protein Kim et al., 2009

CX070113 Starch branching enzyme Kim et al., 2009

Cit.36225.1.S1 Phloem-specific lectin PP2-like protein Albrecht and Bowman, 2008

Cit.9625.1.S1 Glucose-6-phosphate/phosphate translocator Albrecht and Bowman, 2008

Cit.35955.1.S1 Phloem protein 2-B15 (PP2-B15) Albrecht and Bowman, 2008

Cit.11460.1.S1 ZIP5, zinc transporter 5 precursor Albrecht and Bowman, 2012

Cit.940.1.S1 2-oxoglutarate (2OG) and Fe(II)-dependent

oxygenase superfamily protein

Albrecht and Bowman, 2012

Cit.23704.1.S1 CDR1, constitutive disease resistance 1 Albrecht and Bowman, 2012

Cit.28117.1.S1 CDR1, constitutive disease resistance 1 Albrecht and Bowman, 2012

No change related to SC treatment:

CK935883 Disease resistance-responsive protein Kim et al., 2009

B292132 Granule-gound starch synthase Kim et al., 2009

Cit.13437.1.S1 ADP-glucose pyrophosphorylase large subunit Albrecht and Bowman, 2008

Cit.32625.1.S1 Photoassimilate-responding protein (PAR-1a) Albrecht and Bowman, 2008

Internal controls (no change)

citrus 18S rRNA Maul et al. 2007

β- actin Xiao et al. 2011

a, b A stronger band intensity indicates a stronger gene expression

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and middle) of the feeder root crown. Fifty g newlysampled soils were dried at 105 °C for 8 h, weightedand shaked through 2 mm, 1 mm, and 0.3 mm sievesrespectively. Titration methods were used to determinethe N, P, K (used soils through 2 mm sieve) andorganic matter (used soils through 0.3 mm sieve)quantity, while pH (used soils through 2 mm sieve)meter or atomic absorption spectrophotometer wasapplied to measure pH or microelements (Cu, Zn, Fe,and Mn, used soils through 1 mm sieve) (Bao 2000).

Fruit production and quality analyses

All fruits with economic value were collected from each6-year-old SC-treated or non-SC-treated Shatangju trees(Experiment 3) and weighted separately to record theproduction of a single tree. Average weight of individualfruits was calculated by individually weighting 20 ran-domly selected fruits from SC-treated or non-SC-treatedtrees. Soluble solids of mandarin fruits were measured byN-1 α-Hand-held refractometer (ATAGO CO, LTD.)

using fresh juice. Finally, ascorbic acid content weremeasured by 2,6-dichloro-indophenoltitration method(Huang and Liu 2002).

Data analysis

Ct values from Real-time PCR were converted to 16SrDNA concentration (fg μl−1) according to the stan-dard linear regression generated by 16S rDNA plas-mid clone using the TaKaRa Thermal Cycler Dice™Real Time System Software (version 4.01; for TP800).The final DNA concentration data were converted intogenome equivalent copy number/g of citrus tissue(GECN/g) (Duan et al. 2009; Wang et al. 2006) torepresent “Ca. L. asiaticus” titer.

All data were statistically analyzed using SPSS ver.16.0 Software (IBM, Armonk, NY, USA). One-wayANOVA was used to analyze the variance for “Ca. L.asiaticus” titres. The mean separation using least signifi-cant different (LSD) test at 5 % probability level. GLM

T1 T2 T3 T5T4

CK1 CK2 CK3 CK5CK4

a b c d

Fig. 1 A comparison of tree vigour in the 4-year old mandarincitrus trees (Citrus reticulata cultivar Chuntianju) at 10 monthpost treatments (MPT). Photos T1, T2, T3, T4, and T5 show soilconditioner (SC) treatments and photos CK1, CK2, CK3, CK4,

and CK5 show non-SC treatments. Photos a (mature) and b(young) show SC-treatments. Photos c (mature) and d (young)show non-SC treatment

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(General Linear Model) program was conducted to de-termine the significance of difference among fixed factorsof treatment (SC-treated and non-SC-treated), leaf status(mature and young), and age (2-, 3-, and 8-year-old) orbatch (the 1st, 2nd, 3rd and 4th sampling of 4-year-oldtrees). Student-Newman-Keuls test and least significantdifference procedure in multiple comparisons in ANOVAwere applied to analyze the soil nutrient difference. Allgraphs were made using SigmaPlot 12.0 software.

Results

At the beginning of this study, all samples collectedfrom HLB trees were tested positive with nested PCRand had Ct values of 18.29 to 23.44 in real-time PCR,indicating high titres of “Ca. L. asiaticus” in them.There were no significant titre differences betweenyoung and mature leaves of each selected branch (datanot shown).

GE

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/g o

f citr

us ti

ssue

2-Yr old orchard 3-Yr old orchard 8-Yr old orchard

P(Treatment)=0.181P(Leaf stage)=0.768

P(Treatment)=0.072P(Leaf stage)=0.456

P(Treatment)=0.005P(Leaf stage)=0.472

SC treated Non-SC treatedSC treated Non-SC treatedSC treated Non-SC treated

GE

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Fig. 2 Comparison of titre differences of “Ca. L. asiaticus”between soil conditioner (SC) treated and non-treated mandarincitrus (Citrus reticulata cultivar Shatangju) in three orchards

located in Sihui City, Guangdong Province of China at 2 monthspost treatment (MPT). GECN/g: genome equivalent copy number/gof citrus tissue

Table 2 Statistics on variations of “Candidatus Liberibacter asiaticus” titres from HLB affected mandarin citrus trees (Citrus reticulatacultivar Shatangju) of different ages 2 month after treatment with soil conditioner

Source Type III Sum of Squares Df Mean Square F P value

Corrected Model 5.899E+10 11 5.362E+09 2.227 0.049

Intercept 6.808E+10 1 6.808E+10 28.273 0.000

Treatmenta 2.429E+10 1 2.429E+10 10.086 0.004

Tree Ageb 2.176E+10 2 1.088E+10 4.519 0.022

Leaf stagec 1.049E+09 1 1.049E+09 0.436 0.516

Treatment – Tree age 4.700E+09 2 2.350E+09 0.976 0.391

Treatment – Leaf stage 1.740E+09 1 1.740E+09 0.723 0.404

Tree age – Leaf stage 2.342E+09 2 1.171E+09 0.486 0.621

Treatment – Tree age – Leaf state 3.102E+09 2 1.551E+09 0.644 0.534

Error 5.779E+10 24 2.408E+09

Total 1.849E+11 36

Corrected Total 1.168E+11 35

a Treatment: Treated and untreated with soil conditionerb Tree age: 2, 3, and 8 yearc Leaf stage: leaves of young (top) and mature (bottom)

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SC treatment suppressed symptom expression

In the Longmen orchard (Experiment 2), new leavesbegan to develop 1 MPT. Additional healthy lookingmature leaves were seen 3 and 4 MPT. This trendcontinued in the following months. At 10 MPT, typicalHLB symptoms were significantly reduced (Fig. 1,T1–-T5). However, in the non-SC treated trees, HLBseverity increased and tree vigour deteriorated as timeprogressed into autumn and winter. In spring andsummer, new shoots emerged but the leaves weresmaller, chlorotic, or mottling (Fig. 1, CK1–CK5). Aview of young (top) and mature (bottom) leaves inboth SC treated and non-treated trees is shown inFig. 1. Both mature (Fig. 1a) and young (Fig. 1b)

leaves of SC treated trees were healthier-looking thanthose from the non-treated trees (Fig. 1c and d) showingtypical HLB symptoms.

SC treatment was more effective to younger treesthan to older trees

“Ca. L. asiaticus” titres among SC- treated and non-SC-treated citrus trees of three different ages(orchards) in Sihui location (Experiment 1) are sum-marized in Fig. 2. Results of statistical analyses areshown in Table 2. There were significant titre differ-ences between SC and non-SC treatments (P=0.004),suggesting that the use of SC led to the reduction of “Ca.L. asiaticus” titre. In addition, there were significant

4 Month Post Treatment

7 Month Post Treatment 10 Month Post Treatment

1 Month Post Treatment

GE

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/g o

f citr

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GE

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GE

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SC treated Non SC treated SC treated Non SC treated

SC treated Non SC treated SC treated Non SC treated

P(Treatment)=0.639P(Leaf stage)=0.003

P(Treatment)=0.002P(Leaf stage)=0.000

P(Treatment)=0.029P(Leaf stage)=0.081

P(Treatment)=0.925P(Leaf stage)=0.048

Fig. 3 Comparison of titre differences of “Ca. L. asiaticus”between soil conditioner (SC) treated and non-treated mandarincitrus (Citrus reticulata cultivar Shatangju) among four differenttimes (months post treatment, or PMTs) of a growing season in a

4-year old orchard located in Longmen City, Guangdong Prov-ince of China. GECN/g: genome equivalent copy number/g ofcitrus tissue

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differences in “Ca. L. asiaticus” titres among trees ofdifferent ages (P=0.022). Interestingly, there was nosignificant difference associated with leaf stage,i.e. between mature and young leaves (P=0.516).However, in individual orchards, there were significantbacterial titre differences in young leaves of 2-year-oldtrees between the SC and non-SC treatments (Fig. 2).No other significant differences were found in the com-binations of SC treatment * tree age, SC treatment * leafstage, tree age * leaf stage, and SC treatment * tree age *leaf stage.

“Ca. L. asiaticus” titres varied as season progressed

Variations of “Ca. L. asiaticus” titres in the 4-year oldtrees (Experiment 2) in a growing season at 1, 4, 7 and10 MPT are illustrated in Fig. 3. The overall differenceof “Ca. L. asiaticus” titres between SC and non-SCtreated trees had a P value of 0.063 (Table 3). Thiscould be considered to be marginally significant.However, when the data at 10 MPT were not consid-ered, a much higher level of significance (P=0.014)was obtained, suggesting a diminishing effect of SCtreatment at 10 MPT. “Ca. L. asiaticus” titres amongthe four different sampling times (1, 4, 7 and 10 MPT)were significantly different (P<0.001). Unlike the tree

age experiment, the young and mature leaves of 4-yearold trees had significant different “Ca. L. asiaticus”titers (P<0.001). Figure 3 shows “Ca. L. asiaticus”titre differences between young and mature leaves ateach MPT. An important observation was made at 7MPT. There were significant bacterial titre differenceswhen compared to samples at 1 MPT (P<0.001), 4MPT (P=0.024), and 10 MPT (P=0.004), indicating amaximum effect time of SC treatment. Yet at 10 MPT,the differences were not significant (P=0.048 for leafstage and P=0.925 for treatment), providing evidencefor the re-bound of “Ca. L. asiaticus” populations inthe SC-treatment, or the diminishing of SC effects.

SC had effects on changing the physical propertiesand nutrient content of treated soil

Comparisons of the nutritional status between SC- treat-ed and non-treated soil of the Qingyuan orchard(Experiment 3) are shown in Table 4. The significantdifferences are: organic matter (P=0.004), N (P<0.001),P (P<0.001), K (P=0.003) and Mn (P=0.019). In con-trast, there were no significant difference in threemicronutrients, including Cu (P=0.197), Fe (P=0.502),and Zn (P=0.051). The pH values of SC-treated or non-treated soils were not statistical different (P=0.053).

Table 3 Statistics on variations of “Candidatus Liberibacter asiaticus” titres from 4-year-old HLB affected mandarin citrus trees(Citrus reticulata cultivar Shatangju) treated and untreated with soil conditioner during a growing season

Source Type III Sum of Squares df Mean Square F value P value

Corrected Model 1.84389E+12 15 1.22926E+11 4.811 0.000

Intercept 8.3715E+12 1 8.3715E+12 327.651 0.000

Treatmenta 91407122562 1 91407122562 3.578 0.063

Sampling timeb 7.95661E+11 3 2.6522E+11 10.380 0.000

Leaf stagec 7.15376E+11 1 7.15376E+11 27.999 0.000

Treatment – Sampling time 1.31698E+11 3 43899281147 1.718 0.172

Treatment – Leaf stage 68525490903 1 68525490903 2.682 0.106

Sampling time – Leaf stage 34631176747 3 11543725582 0.452 0.717

Treatment – Sampling time – Leaf stage 6592798800 3 2197599600 0.086 0.967

Error 1.6352E+12 64 25550055337

Total 1.18506E+13 80

Corrected Total 3.4791E+12 79

a Treatment: Treated and untreated with soil conditionerb Sampling time: 1, 4, 7, and 10 month post treatmentc Leaf stage: leaves of young (top) and mature (bottom)

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SC treatment affected fruit yields and qualitybut not soil microbial diversity

Both total fruit yield per tree and average weight ofindividual fruit from SC- treated trees in the 6-year-oldShatangju orchard (Experiment 3) were significantlyhigher than those from the non-treated trees (P<0.001)(Table 5). Both soluble solids concentration (P=0.03)and ascorbic acid content (P=0.005) were significantlyhigher than those of the non-SC treated trees (Table 5).Microbial plate counts on SC- treated and non-SCtreated soils showed a slight increase of actinomycetes(P=0.068) in SC-treated soils, and no significant dif-ferences in bacteria (P=0.656) and fungi (P=0.148).

Expression profile of selected candidate genes

The expression profiles of the 25 selected genes in SC-and non-SC-treated citrus are presented in Table 1.Among them, eight genes were up-regulated in SC-treated trees. These include five defence response genesa vitamin C defective 2 protein (VTC2, Cit.8866.1.S1),

a plant defensin (PDF2.2, Cit.2831.1.S1) belonging tothe PR-12 family, a pathogenesis-related protein PR-1precursor (CF653559), a putative cell death associatedprotein (CX045772) and a transcription factor of Mybprotein which is a key factor in the regulatory networkscontrolling development, metabolism and response tobiotic and abiotic stresses (Dubos et al. 2010). In con-trast, 11 genes were down-regulated, including fivestarch synthesis related genes (starch branching enzyme,plant glycogenin-like starch initiation protein, β-amylase, phloem-specific lectin PP2-like protein, andglucose-6-phosphate/phosphate translocator). Variationsin the expression of other six selected genes includingthe two internal controls were not observed.

Discussion

Management of HLB based on nutritional approacheshas been controversial (Zhang et al. 2007; Pustika etal. 2008; Ahmad et al. 2011; Razi et al. 2011; Spann etal. 2011; Xia et al. 2011). In this study, we observedthe less severe HLB symptoms, reduced “Ca. L.asiaticus” titres, and increased fruit yield and qualityin HLB trees after SC treatment. In addition, SCtreatment was associated with the increase of N, P,K, Mn and organic matters in soil. SC treatment wasmore effective in reducing the severity of HLB inyoung trees than in old trees. The lack of no responseto SC treatment of the 8-year-old HLB-affected treesmay be related to the severe damage of root system(Pustika et al. 2008). The success of SC treatment formitigating HLB may explain the observation that, his-torically and empirically, citrus growers in Guangdongroutinely used manure or compost for orchard manage-ment, not only to improve tree growth, but also toseemingly prolong tree life even in the presence ofHLB symptoms (Xia et al. 2011).

Table 5 Yield and quality of fruit harvested from 6-year-old Shatangju trees treated with soil conditioner (SC) and trees of untreatedcontrol

Treatment Production (kg/tree) average weight ofindividual fruit (g)

Soluble solids contents(%) Ascorbic acidcontent(mg/100 g)

SC 62.48±0.51a 52.31±2.00a 13.50±0.12a 23.42±0.42a

Non-SC 36.60±0.51b 45.58±1.47b 11.3±0.08b 20.20±1.00b

Values with different letters within each column means significant difference (P<0.05)

Table 4 Nutrient and organic matter status of soil conditioner-treated and -untreated soil of the 6-year-old Shatangju orchard2 months post treatment

Nutrients Treated Non-treated P value

PH 4.77±0.14 4.99±0.17 0.053

Organic matters 28.48±1.86 26.60±0.78 0.004

N 146.26±7.80 127.03±6.76 <0.001

P 159.69±14.13 103.35±13.41 <0.001

K 272.86±18.11 213.45±47.03 0.003

Cu 3.31±0.36 2.73±0.55 0.197

Fe 111.57±3.16 108.43±6.65 0.502

Mn 25.93±7.37 9.26±1.84 0.019

Zn 14.00±2.53 8.73±2.14 0.051

Eur J Plant Pathol

We observed various types of gene expression incitrus hosts related to SC treatments. One interestingobservation is the down-regulation of the five starchsynthesis related genes after SC treatment. Starch syn-thesis genes were related to callose deposition to plugthe sieve pores in HLB-affected plants. With the ex-ception of the phloem-specific lectin PP2-like protein,all of the four starch synthesis genes were up-regulatedafter inoculation of “Ca. L. asiaticus” (Albrecht andBowman 2008; Kim et al. 2009). The molecular mech-anisms of how SC treatment improves the performanceof HLB affected trees deserve more studies. The datapresented in Table 1 could serve as an initial point toinvestigate SC effects on HLB in the future.

It should be noted that positive effects of SC onHLB trees could only be achieved by following upwith appropriate cultural management and pest con-trol. In the 8-year-old orchard of Experiment 1, thegrower abandoned the orchard after 3 MPT. Continuedobservation showed that the psyllid population quicklyincreased, especially on the new flushes of the SC-treated trees; weed growth went out of control; andsimilarly severe HLB symptoms (Zn-deficiency-likeon young leaves and blotchy mottle on mature leaves)were observed on both the SC- and non-SC treated treesin the following 3months. As shown fromExperiment 2(titre vs. growth season), “Ca. L. asiaticus” titres in SC-treated trees increased to the same levels as those in thenon-SC-treat trees 10 MPT, even with the best culturalmanagement.

A previous study reported an inconsequential effectof enhanced nutritional programs on HLB control,fruit quality, bacterial titre and disease progress inFlorida (Gottwald et al. 2012). Our preliminary studyseems to show a positive effect of nutrient manage-ment or SC treatment on mandarin production inGuangdong, China. However, our field experimentswere made only for 1 year, and whether the effect ofthe SC can be reproducible over years remains to betested. Further, recovery of HLB trees was observedonly for a few months, indicating the effects are onlytemporary. How to optimize the use of SC in combi-nation with other HLB management measures remainsto be investigated in the future.

In conclusion, preliminary data from this studyshow that SC treatment has positive effects on theHLB affected mandarins (cultivars Shatangju andChuntianju) in Guangdong, China. These includedimproved tree growth with reduced HLB symptoms,

lower “Ca. L. asiaticus” titres in less than 10 months,increased fruit yield and improved fruit quality.Apparently, SC treatment could not eliminate “Ca. L.asiaticus”. SC treatment may induce a defence-response reaction and affect HLB-mediated physio-logical and biochemical process.

Acknowledgments The authors acknowledge the ZaoqingSouth ChinaRun-zhe-xin-ye Biotechnology Limited Liability Com-pany for providing the SC. We appreciate the financial supports ofPublic service sectors (agriculture) special project (Grant No.201003067) from Ministry of Agriculture of the People's Republicof China, National Natural Science Fund (Grant No. 31201480)fromNational Natural Science Foundation of China and production-study-research combination project (Grant No. 2012B091100483)from Ministry of Education of Guangdong Province.

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