Plant and Soil

, Volume 357, Issue 1, pp 245–257

Biochar mediates systemic response of strawberry to foliar fungal pathogens

Authors

  • Yael Meller Harel
    • Department of Plant Pathology and Weed Research, Institute of Plant Protection, The Volcani CenterAgricultural Research Organization
    • Department of Plant Pathology and Weed Research, Institute of Plant Protection, The Volcani CenterAgricultural Research Organization
  • Dalia Rav-David
    • Department of Plant Pathology and Weed Research, Institute of Plant Protection, The Volcani CenterAgricultural Research Organization
  • Menachem Borenstein
    • Department of Plant Pathology and Weed Research, Institute of Plant Protection, The Volcani CenterAgricultural Research Organization
  • Ran Shulchani
    • Department of Plant Pathology and Weed Research, Institute of Plant Protection, The Volcani CenterAgricultural Research Organization
  • Beni Lew
    • Department of Growing, Production and Environmental Engineering, Institute of Agricultural Engineering, The Volcani CenterAgricultural Research Organization
  • Ellen R. Graber
    • Department of Soil Chemistry, Plant Nutrition and Microbiology, Institute of Soil, Water and Environmental Sciences, The Volcani CenterAgricultural Research Organization
Regular Article

DOI: 10.1007/s11104-012-1129-3

Cite this article as:
Meller Harel, Y., Elad, Y., Rav-David, D. et al. Plant Soil (2012) 357: 245. doi:10.1007/s11104-012-1129-3

Abstract

Background and Aims

Various biochars added to soil have been shown to improve plant performance. Moreover, a wood biochar was found to induce tomato and pepper plant systemic resistance to two foliar fungal pathogens. The aim of this study was to explore the ability of wood biochar and greenhouse waste biochar to induce systemic resistance in strawberry plants against Botrytis cinerea, Colletotrichum acutatum and Podosphaera apahanis, and to examine at the molecular level some of their impacts on plant defense mechanisms.

Methods

Disease development tests on plants grown on 1 or 3% biochar-amended potting mixture, and quantification of relative expression of 5 plant defense-related genes (FaPR1, Faolp2, Fra a3, Falox, and FaWRKY1) by real-time PCR were carried out.

Results

Biochar addition to the potting medium of strawberry plants suppressed diseases caused by the three fungi, which have very different infection strategies. This suggests that biochar stimulated a range of general defense pathways, as confirmed by results of qPCR study of defense-related gene expression. Furthermore, primed-state of defense-related gene expression was observed upon infection by B. cinerea and P. aphanis.

Conclusion

The ability of biochar amendment to promote transcriptional changes along different plant defense pathways probably contributes to its broad spectrum capacity for disease suppression.

Keywords

Biotic stressInduced systemic resistancePlant diseasePrimingSystemic acquired resistanceSystemic resistance

Abbreviations

qPCR

real time quantitative PCR

RT

reverse transcription

IR

induced resistance

ISR

induced systemic resistance

SAR

systemic acquired resistance

HR

hypersensitive reaction

PR

pathogenesis related proteins

SA

salicylic acid

PGPR

plant growth-promoting rhizobacteria

PGPF

plant growth-promoting fungi

ET

ethylene

JA

jasmonic acid

MeJA

methyl jasmonate

CW

biochar produced from citrus wood

GHW-450

biochar produced from greenhouse waste at 450°C

AUDPC

area under the disease progress curve

Ct

cycle threshold

Introduction

Scientific, commercial and political interest in using biochar (solid co-product of biomass pyrolysis) as a soil amendment for purposes of sequestering carbon and enhancing soil fertility has been growing apace in recent years. To now, a number of field and pot trials have shown that addition of different biochars to soil can enhance productivity of various crops (Asai et al. 2009; Graber et al. 2010; Major et al. 2010; Vaccari et al. 2011). A host of different processes operating singly, in tandem or synergistically have been purported to be responsible for the observed improvements in crop performance. Among other effects, biochar addition to soil leads to improvement in soil pH, higher soil cation exchange capacity, increased soil retention of nutrients and water, greater mycorrhizal competence, and improved soil physical characteristics (Atkinson et al. 2010). Biochar can also add nutrients to the soil system (Hossain et al. 2011; Silber et al. 2010; Uzoma et al. 2011).

In addition to these effects, it was recently discovered that amending soil with biochar can potentiate system-wide plant defensive responses against disease. In the first report of its kind, it was found that the severity of the fungal foliar diseases caused by Botrytis cinerea Pers.:Fr. and Oidiopsis sicula Scalia (identified therein as the teleomorph: Leveillula taurica (Lév.) Arnaud) in tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum L.) was significantly reduced in biochar-amended potting medium (Elad et al. 2010). Pepper plants were additionally protected against damages from the broad mite pest (Polyphagotarsonemus latus Banks). The fact that the biochar location during all stages of plant development was spatially separated from the site of infection indicated that direct toxicity towards the causal agents was not involved, and pointed to an induced systemic response of the plant against the biotic stresses.

In general, two major pathways leading to system-wide induced resistance (IR) in plants have been described. Systemic acquired resistance (SAR), which usually starts with a hypersensitive reaction (HR) leading to local necrosis, involves synthesis of pathogenesis-related (PR) proteins and is mediated by the phytohormone salicylic acid (SA). Induced systemic resistance (ISR), commonly triggered by plant growth-promoting rhizobacteria (PGPR) and fungi (PGPF), depends on the phytohormones ethylene (ET), jasmonic acid (JA) and methyl jasmonate (MeJA) (Vallad and Goodman 2004). Thus far, no studies have investigated the plant defense pathways induced by biochar.

Considering the widespread consumer pressure to reduce the use of chemical pesticides in agriculture, and the growing interest in utilizing biochar in soil for carbon sequestration purposes, the development of agricultural markets for biochar as a disease control agent could help promote the adoption of biomass pyrolysis as one of the tools in the arsenal for combating climate change. To do this, it is necessary to explore the potential of different biochars to induce plant systemic resistance to disease in more crop systems, as well as to make headway in understanding the mechanisms responsible for the induction. This is the aim of the present study, which examines the role of two biochars in IR in strawberry plants (Fragaria × ananassa Duch.) against the foliar fungal pathogens B. cinerea (causal agent of gray mold), Podosphaera apahanis (Wallr.) Braun & Takam (responsible for powdery mildew in strawberry), and Colletotrichum acutatum Simmonds (one of the pathogens responsible for anthracnose). These fungi have very different infection strategies: necrotrophic (B. cinerea), hemi-biotrophic (C. acutatum), and biotrophic (P. apahanis). In addition to evaluating whether biochar induces systemic resistance against these diseases in strawberry, the expression of five strawberry defense-related genes in leaves as affected by biochar in the root zone and/or infection by the pathogens B. cinerea and P. apahanis, was monitored using quantitative real time PCR (qPCR). For the first time, the results presented herein attempt to shed some light on molecular mechanisms which may be involved in biochar-mediated systemic responses of plants to phytopathogens.

Materials and methods

Biochar and plant growth medium

Biochar from two feedstocks was used throughout the research: (i) biochar produced from citrus wood (CW) in a traditional charcoal pit, previously described (Elad et al. 2010; Graber et al. 2010) and (ii) biochar produced from greenhouse wastes (mainly pepper plant wastes), GHW-450, in an in-house pyrolysis reactor operated in indirect retort mode at a highest treatment temperature of 450°C. Biochars were ground into a powder of <0.5 mm particles and stored in sealed containers. pH and electrical conductivity (EC) of 1:20 w:v biochar:double distilled water suspensions were determined to be: (i) CW: pH of 7.6, EC of 1.6 dS m−1; (ii) GHW-450: pH of 9.7, EC of 9.5 dS m−1. The high salt content of the GHW-450 biochar is a residue of salts taken up by the pepper plants during their growth (grown on slightly saline water); these salts are quickly rinsed out of the potting media as a result of the frequent irrigations. Biochar at 1 or 3% (w:w) was mixed with potting mixtures consisting of coconut fiber:peat (unsorted to 8 mm; 1:1 vol:vol) or tuff:peat (7:3 vol:vol).

Plants and fertigation regime

Strawberry plants (cv. Yael) were obtained from a commercial nursery at 50 to 80 days after rooting and transplanted into one L-pots. Plants were fertigated proportionally with drippers two to three times per day with 5:3:8 NPK fertilizer (irrigation water was planned to have total N, P and K concentrations of 120, 30 and 150 mg L−1, respectively; EC 2.2 dS m−1), allowing for 25–50% drainage. Plants were maintained at 20 to 30°C in a pest- and disease-free greenhouse and then transferred to an area where diseases were allowed to develop following inoculation of intact leaves as described below. Alternatively, stolons were grown out of mother plants, and the produced daughter plants were rooted in a peat media in a humid chamber for 35 days. Fully rooted daughter plants were moved to the proper growth mix and fertigated as described above. Daughter plants were obtained by rooting stolons from the mother plants in a peat media in a humid chamber for 35 days. Fully rooted daughter plants were then transplanted to the potting mixtures as above.

Fungal pathogens, inoculation and disease evaluation

Botrytis cinerea

Strawberry plants were grown on coconut fiber:peat mix amended with 0, 1 or 3% (w:w) GHW-450 or CW biochar for 11 to 90 days before disease inoculation. B. cinerea (isolate BcI16) (Swartzberg et al. 2008) was cultured on potato dextrose agar (PDA) (Difco Laboratories, Detroit) in Petri dishes incubated at 20°C for 4 days. Agar disks (2 mm) containing pathogen mycelium were cut out from the colony edge and placed, mycelium side down, on the surface of 3 young fully expanded strawberry leaflets, with 3–6 disks per leaf. The inoculated plants were sprayed with water and maintained in humidity chamber at 20°C. The evaluation of gray mold severity was followed for 9 days by measuring the diameter of the lesion from which rot area was calculated.

Colletotrichum acutatum

Strawberry plants were grown on tuff:peat mix or coconut fiber:peat mix amended with 0, 1 or 3% (w:w) GHW-450 biochar for 3.5 months to 6 months before disease inoculation. C. acutatum conidial suspension was obtained from the laboratory of Dr. Stanley Freeman at the Volcani Center, Bet Dagan and used to inoculate a PDA plate for 5 days at 25°C. Conidia were scrapped out in sterile distilled water flooding the plates. Concentration of filtered conidial suspension was determined using a haemocytometer and adjusted to 104 mL−1. Conidial suspension (5 mL plant−1) was sprayed on foliage. Plants were covered in plastic bags for 4 days and maintained in a greenhouse chamber at 28°C, high humidity and 8 hours of light per day. Anthracnose incidence was followed for 35 days by counting lesions on the aerial parts of the plants.

Podosphaera aphanis

Strawberry plants were grown on tuff:peat mix amended with 0, 1 or 3% (w:w) CW biochar or coconut fiber:peat mix amended with 0, 1 or 3% (w:w) GHW-450 biochar for 11 days to 9 months before disease inoculation. P. aphanis was obtained from naturally infected leaves (cv. Tamar, susceptible to disease, Sharon region, Israel) and maintained on strawberry plants grown in a growth chamber at 22 to 25°C (day) and 15 to 18°C (night). Concentration of conidial suspension obtained from infected leaves was determined on a haemocytometer and adjusted to 104 mL−1. Conidial suspension (5 mL plant−1) was sprayed on foliage within 10 to 15 min of the initial conidia collection. Disease severity (percentage of infected area) was rated several times over the course of 35 days on three designated leaves per plant.

Experimental design and statistical analysis

Greenhouse and growth chamber experiments were carried out in two to three trials, with 6 replicate plants per treatment, per trial. Data for representative experiments are presented. Areas under the disease progress curves (AUDPC values) were calculated (Foolad et al. 2000) for disease severity and incidence. Data in percentages were arcsin-transformed before further analysis. Percent reduction in disease severity (% Reduction) was calculated as:
$$ \% \, Reduction = - \left( {\frac{{AUDP{C_{{BC}}} * 100}}{{AUDP{C_{{Control}}}}}} \right) $$
where BC refers to treatment where biochar was added to the potting mix, and control refers to the treatment where no biochar was added to the potting mix. Treatments and replicates were fully randomized in the greenhouse and growth chambers. Disease incidence and AUDPC data were analyzed using one-way analysis of variance (ANOVA) for significance and Fisher’s protected LSD test for mean separation (P ≤ 0.05). Standard errors of the means were also calculated. The standard errors are marked with error bars in the figures and stated in the tables. Statistical analysis was done using the R version 2.10.1 software (http://www.r-project.org).

Molecular analysis

Genes studied: The studied defense-related strawberry genes were FaPR1 (GenBank AB462752.1), Faolp2 (GenBank DQ325524.1) and Fra a3 (GenBank GQ148819), which encode PR proteins, Falox (GenBank AJ578035), which encodes a lipoxygenase enzyme, and FaWRKY1 (GenBank EU727547), coding for a trans-acting factor from the WRKY family.

RNA extraction: RNA was extracted from young fully expanded strawberry leaves using the procedure described previously (Asif et al. 2000). Leaf tissue was pooled from 3 independent biological replicates to reduce noise linked to variation in experimental conditions. The RNA concentration was measured on an ND-1000 NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and sample purity was estimated by the ratio of absorbance at 260 nm to absorbance at 280 nm.

cDNA synthesis: RT was performed in two to three replicates on 800 ng of DNAse I-treated total RNA (Applied Biosystems/Ambion, Austin, TX, USA) using the M-MLV reverse transcriptase (Promega, Corporation Madison, WI, USA), and cDNA products were pooled to reduce noise linked to RT efficiency.

qPCR analysis: qPCR reactions were carried out on a Rotor-Gene Q 6000 (Qiagen, Hilden Germany) and results were analyzed using Rotor-Gene 6000 software. Each PCR amplification was performed in duplicate in a 15 μl reaction mixture consisting of 7.5 μl of Absolute Blue SYBR Green ROX qPCR Master Mix 2X (ABgene, Abgene Ltd, Epsom, United Kingdom), 1 μl each of the forward and reverse primers (3 μM), 4 μl of cDNA template, and 1.5 μl of PCR-grade water. The cycling conditions consisted of 15 min of pre-incubation at 95°C followed by 40 cycles of 10 s at 95°C, 15 s at 60°C and 20 s at 72°C. Amplification of one product only was confirmed by melt curve analysis (72 to 95°C). FaGAPDH (accession number AB363963) and a 150-bp specific fragment of an interspacer 18S–26S RNA (Moyano et al. 2004) were used for normalization of gene expression. The primers used for amplification of the genes of interest were designed with the Primer 3 program (http://fokker.wi.mit.edu/primer3/) and synthesized (Integrated DNA Technologies, Inc, Skokie, IL USA). Their sequences are outlined in Table 1. Relative gene expression levels in the treated plants compared to untreated and un-inoculated plants illustrated in the figures below was calculated with the formula 2-ΔΔCt , using the ΔΔ Ct method (Livak and Schmittgen 2001), where ΔCt = Ct specific gene- Ct normalizer and ΔΔ Ct = ΔCt—arbitrary constant (the highest ΔΔ Ct, (Shoresh et al. 2005)). Fold-induction of gene expression as given in the text was calculated as the quotient of average relative gene expression in the treated sample and average relative gene expression in the control (no biochar and no disease) sample.
Table 1

Primers used in the qPCR experiments. Sequences are given in the 5′ to 3′ orientation

Gene name

GenBank accession number or reference

Forward Primer (5′ to 3′)

Reverse Primer (5′ to 3′)

FaGADPH

AB363963

gaa gac tgt tga tgg acc atc

tgt cac caa tga agt cgg ttg

Interspacer 18S–26S RNA

(Moyano et al. 2004)

acc gtt gat tcg cac aat tgg tca tcg

tac tgc ggg tcg gca atc gga cg

FaPR1

AB462752.1

aca tgg gat gcc aat cta gc

cca cag gtt cac agc aga tg

Faolp2

DQ325524.1

aat tct ggg agc tgt caa cc

ggc aga aca caa ccc tat ag

Fra a3

GQ148819

ggg aga tgc tat cgg aga ca

ctt tcc ttt ccg gcc tta ac

Falox

AJ578035

agg caa atc ctc atc aat gc

gct cgg tga ata ccc agt gt

FaWRKY1

EU727547

cat gca ttc tca tcc cat tg

acg gac agc agg cat atg ta

Results

Effect of biochar on disease severity

Effect of CW and GHW-450 biochars on gray mold, anthracnose and powdery mildew

Both tested biochars, one produced from citrus wood (CW) and one from greenhouse waste (GHW-450), were found to lower significantly gray mold disease severity on plants grown in coconut:peat potting mix for 25 days prior to the disease challenge, relative to the control plants (Fig. 1). All three biochar treatments (1% GHW-450, 3% GHW-450, 3% CW) resulted in the same extent of reduced disease severity, as quantified by the area under the disease progression curves (AUDPC). Nine days following the pathogen challenge, AUDPC values (average ± SE) were 416 ± 50.5, 158 ± 33.0, 110 ± 32.0, and 118 ± 24.3 mm2 × days in the 0% biochar, 1% GHW-450, 3% GHW-450 and 3% CW biochar treatments, respectively (biochar treatments significantly different from control, P ≤ 0.05). GHW-450 biochar was also found to significantly reduce disease severity of C. acutatum on attached strawberry leaves when applied at amendment rate of 3% for 105 days in tuff:peat mix (Fig. 2). The calculated AUDPC of whole-plant disease severity during 35 days of infection was significantly reduced by 49% from 159 ± 27.4 to 81 ± 12.3 no. × days. No effect of GHW-450 at the amendment rate of 1% in tuff:peat mix could be seen on severity of C. acutatum (Fig. 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs11104-012-1129-3/MediaObjects/11104_2012_1129_Fig1_HTML.gif
Fig. 1

Effect of biochar amendment on development of gray mold on attached strawberry leaves. Plants were grown in coconut:peat potting medium amended with either greenhouse waste (GHW-450) or citrus wood (CW) biochar for 25 days. Botrytis cinerea infection is presented as rot area through nine days. Bars represent the standard error of the mean of six replicates. Data points labeled by a common letter are not significantly different according to Fisher’s protected LSD test. Pictures (bottom) represent gray mold on strawberry leaves 9 days after B. cinerea infection from treatments of no biochar (left), 1% GHW-450 (middle), and 3% GHW-450 (right)

https://static-content.springer.com/image/art%3A10.1007%2Fs11104-012-1129-3/MediaObjects/11104_2012_1129_Fig2_HTML.gif
Fig. 2

Effect of biochar amendment on development of anthranose on strawberry plants. Plants were grown in coconut tuff:peat potting mixture amended with greenhouse waste (GHW-450) biochar for 105 days. Colletotrichum acutatum infection is expressed as incidence of infection foci 35 days following infection. Bars represent the standard error of the mean of six replicates. Data points labeled by a common letter are not significantly different according to Fisher’s protected LSD test. Pictures (bottom) represent anthracnose infection on strawberry plants from treatments of no biochar (upper pictures), and 3% citrus wood (CW) (lower pictures) as viewed from the side (left) and from above (right)

CW biochar amendment at a level of 3% was found to protect strawberry plants against P. aphanis as well (Fig. 3). Disease severity on attached leaves was significantly reduced in plants grown for 147 days in tuff:peat growing mix amended with 3% CW biochar. The calculated AUDPC of whole-plant disease severity during 35 days of infection was significantly reduced by 68 ± 13.9%, from 942 ± 47.2 to 304 ± 38.1% × days. No significant effect could be seen for 1% CW amendment (Fig. 3).
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Fig. 3

Effect of biochar amendment on development of powdery mildew on strawberry leaves. Plants were grown in coconut fiber:peat potting mixture amended with citrus wood (CW) biochar for nine months. Podosphaera aphanis infection severity is presented as percent leaf coverage over 35 days. Bars represent the standard error of the mean of six replicates. Data points labeled by a common letter are not significantly different according to Fisher’s protected LSD test. Pictures (bottom) represent powdery mildew on strawberry leaves from treatments of no biochar (left), and 3% CW (right) 28 days after infection

Effect of exposure duration, growth mix and amendment rate on biochar impact on induced resistance

While plants grown in 1% GHW-450 amended coconut:peat potting mix for 25 days prior to B. cinerea challenge experienced a 62 ± 15.1% reduction in disease severity, no effect of biochar on B. cinerea severity could be observed on plantlets grown in the same 1% biochar-potting mix for ten days only (Table 2). However, amendment at a higher rate of biochar (3% GHW-450) reduced B. cinerea severity by similar extents in both mature and young plants (74 ± 23.3% reduction in the case of mature plants grown in biochar amended potting mix for 25 days and 53 ± 17.4% reduction in the case of young plantlets grown in biochar-amended potting mix for ten days) (Table 2).

The type of potting mix (coconut fiber:peat or tuff:peat) had a significant influence on the extent of biochar impact against anthracnose (C. acutatum) (Table 3). Plants grown for 105 days in coconut fiber:peat mix with 1% GHW-450 exhibited a 50 ± 13.2% reduction in anthracnose severity, as calculated from the AUDPC values 35 days following the pathogen challenge (Table 3). No such effect was observed for plants grown for the same period in tuff:peat mix amended with the same biochar at the same 1% level (Table 3). Yet, amendment with 3% GHW-450 reduced anthracnose severity in plants grown in both potting mixes by similar rates (39 ± 8.9% in the case of coconut fiber:peat mix and 49 ± 11.2% in the case of tuff:peat mix; Table 3). Finally, the absence of effect of 1% biochar on P. aphanis reduction (Fig. 3) was systematically observed in other experiments, independent of biochar type, plant growing period, potting mix, plant age or type of infection (spontaneous or purposely induced) (data not shown).
Table 2

Effect of plant age and exposure duration on induced resistance to gray mold by GHW-450 biochar amendment

GHW-450 (%)A

Mature plants grown for 25 days in biochar

Daughter plants grown for 11 days in biochar

0

416B ± 50.5 aC

80 ± 17.0 a

1

157 ± 33.0 b

81 ± 18.6 a

3

110 ± 32.0 b

38 ± 9.6 b

A Plants were grown in coconut:peat potting mixture amended with GHW-450 biochar

BB. cinerea infection is expressed as area under disease pressure curves (AUDPC ± SE) for nine days following infection

C Data values in each column labeled by a common letter are not significantly different (P ≤ 0.05) according to Fisher’s protected LSD test

Table 3

Effect of growth medium on reduction of anthracnose on strawberry leaves by GHW-450 biochar amendment

GHW-450 (%)A

coconut:peat mix

tuff:peat mix

0

207B ± 41.3 aC

159 ± 27.4 a

1

104 ± 18.2 b

165 ± 16.1 a

3

127 ± 14.2 b

81 ± 12.2 b

A Fifty days old plants were grown in potting mixtures amended with GHW-450 biochar for 105 days

BColletotrichum acutatum infection is expressed as area under disease pressure curves (AUDPC ± SE) for 35 days following infection

C Data values in each column labeled by a common letter are not significantly different (P ≤ 0.05) according to Fisher’s protected LSD test

Expression of defense-related genes

Pathogen effect

The effect of two pathogens (B. cinerea and P. aphanis) and the two biochars on the expression of five defense-related genes (FaPR1, Faolp2, Fra a3, Falox, and FaWRKY1) in strawberry leaves was quantified by qPCR. Pathogen and biochar effects were examined both individually and in combination.

As anticipated, inoculation of the strawberry plants with each of the pathogens induced a plant response in terms of increased expression of defense-related genes as compared with the non-infected plants. Seven days following plant inoculation with B. cinerea, a significant induction of the expression of all five genes was observed: FaPR1 four-fold, Faolp2 15-fold, Fra a3 22-fold, Falox 68-fold, and FaWRKY1 106-fold (Fig. 4). Twenty-eight days following inoculation of plants with P. aphanis, expression of FaPR1was increased five-fold, Faolp2 72-fold and Fra a3 five-fold (Fig. 5). No effect of P. aphanis on Falox or FaWRKY1 expression was found (Fig. 5).
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Fig. 4

Effect of Botrytis cinerea infection (7 days post inoculation) on expression of defense related genes. Expression of FaPR1, Faolp2, Fra a3, FaLox and FaWRKY1 were analyzed in plants grown for 25 days on coconut fiber:tuff mix in the absence (-) or presence (+) of infection by B. cinerea. Total RNA was isolated from leaves and subjected to qPCR analysis. Fold-change is expressed relative to non-infected control plants. Mean level of relative expression and standard errors from five qPCR experiments are presented

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Fig. 5

Effect of Podosphaera aphanis infection (28 days after inoculation) on expression of defense related genes. Expression of FaPR1, Faolp2, Fra a3, FaLox and FaWRKY1 were analyzed in plants grown for nine months on peat:tuff in the absence (-) or presence (+) of infection by P. aphanis. Total RNA was isolated and subjected to qPCR analysis. Fold-change is expressed relative to non-infected control plants. Mean level of relative expression and standard errors from five qPCR experiments are presented

Biochar effect

The effect of biochar addition per se on the expression of the five defense-related genes in strawberry leaves was also quantified by qPCR and compared to plants grown with no biochar. One time point was evaluated for each biochar: after 25 days of plant growth in the GHW-450 amended growing mix (Fig. 6a), and after nine months of plant growth in the CW amended growing mix (Fig. 6b). The most sensitive gene in both instances was Faolp2, which exhibited extreme induced rate of expression in plants grown on 3% GHW-450 for 25 days (Fig. 6a, 107-fold induction) or after nine months on 1% CW (Fig. 6b, 110-fold induction). In contrast, expression of FaPR1 was at the most induced four-fold in plants grown in 3% CW (Fig. 6b), while its expression remained constant otherwise (Fig. 6a). Finally, expression of Fra a3, FaLox and FaWRKY1 were mildly to moderately induced in all cases (two to four fold (Fig. 6a) and two to 12-fold (Fig. 6b)).
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Fig. 6

Effect of biochar amendments on expression of FaPR1, Faolp2, Fra a3, FaLox and FaWRKY1 defense related genes in plants grown in coconut fiber:peat potting mix amended with (a) greenhouse waste (GHW-450) biochar, and (b) citrus wood (CW) biochar at 1 or 3% by weight. Total RNA was isolated from leaves after the plants grew for 25 days (a) or nine months (b), and subjected to qPCR analysis. Fold-change is expressed relative to plants grown without biochar amendment. Mean level of relative expression and standard errors from five qPCR experiments are presented

Combined pathogen and biochar effect on gene expression

For the case of combined pathogen and biochar, there was a general increase in transcriptional up-regulation of Faolp2, Fra a3, Falox and FaWRKY1 seven days following infection by B. cinerea in plants grown for 25 days on 1% GHW-450 biochar and compared with non-infected plants grown with no biochar (Fig. 7). The induction of Faolp2 expression was stronger in the combined presence of biochar and pathogen than that promoted by the pathogen alone (30-fold in Fig. 7 compared with 15.5-fold in Fig. 4). In contrast, in plants growing in 3% GHW-450 biochar, there was a marked decrease of induction of Faolp2, Fra a3 and FaWRKY, seven days post-infection (Fig. 7). Finally, transcriptional up-regulation of FaPR1 expression was unchanged upon B. cinerea infection at both biochar levels (Fig. 7).
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Fig. 7

Effect of Botrytis cinerea infection (7 days post inoculation) on expression of defense-related genes on plants grown for 25 days on coconut fiber:tuff mix amended with either 1 or 3% greenhouse waste biochar (GHW-450). Expression of FaPR1, Faolp2, Fra a3, FaLox and FaWRKY1 were analyzed in plants in the absence (-) or presence (+) of infection by B. cinerea. Total RNA was isolated from leaves and subjected to qPCR analysis. Fold-change is expressed relative to non-infected plants grown without biochar. Mean level of relative expression and standard errors from five qPCR experiments are presented

Twenty eight days following P. aphanis infection of plants which had grown for nine months in the potting medium amended with CW biochar, expressions of the genes of interest were all inhibited in plants grown on 1% biochar, and all increased in plants grown on 3% biochar (Fig. 8). In plants grown on 1% CW biochar, expression of Faolp2 (110- to 40-fold induction) was particularly inhibited (Fig. 8), and in plants grown on 3% CW biochar, expression of Falox (12- to 40-fold induction) was greatly increased (Fig. 8). Notably, the induction of expression of Falox was much greater than the one promoted by the infection in absence of biochar (48-fold, Fig. 8, compared with 1.8, Fig. 5). In the case of FaPR1 expression, powdery mildew infection led to a 2.5-fold increase, independent of biochar concentration (Figs. 5; 8).
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Fig. 8

Effect of Podosphaera aphanis infection (28 days after inoculation) on expression of defense-related genes on plants grown for nine months on peat:tuff mix amended with either 1 or 3% citrus wood biochar (CW). Expression of FaPR1, Faolp2, Fra a3, FaLox and FaWRKY1 were analyzed in the absence (-) or presence (+) of infection by P. aphanis. Total RNA was isolated and subjected to qPCR analysis. Fold-change is expressed relative to non-infected control plants grown without biochar. Mean level of relative expression and standard errors from five qPCR experiments are presented

Discussion

The spatial separation between the diseases and the control agent points to systemic induced resistance in strawberry plants by biochar amendments towards these foliar pathogens, as was recently described for biochar amendment of tomato and pepper plants (Elad et al. 2010). Systemic resistance induced in strawberry plants by different biological and chemical agents has been previously reported. Biocontrol agents, including Trichoderma spp. have been shown to control powdery mildew, gray mold and anthracnose (De Cal et al. 2008; Freeman et al. 2004; Pertot et al. 2008) while Bion, a functional analogue of SA, has been shown to improve resistance to powdery mildew and gray mold (Hukkanen et al. 2007; Joyce and Terry 2000). Herein we found that two different biochars added to the potting medium of strawberry plants reduced the severity of three foliar diseases caused by fungi having very different infection strategies: necrotrophic (B. cinerea), hemi-biotrophic (C. acutatum), and biotrophic (P. apahanis). Plant defense mechanisms against pathogen attacks are usually tailored differently according to the biological strategy of the pathogen (Glazebrook 2005). The fact that biochar amendment reduced the susceptibility to fungi with these different infection strategies is notable, and suggests that biochar stimulated a range of general defense pathways in the strawberry plants.

In Elad et al. (2010), 1 and 3% amendment rates were generally equally efficient against necrotrophic and biotrophic diseases. Here, in the case of strawberry plants, 1% biochar was not effective against gray mold when young plants were exposed to the biochar for a short time. Nor was 1% biochar effective against anthracnose in tuff:peat potting mix, although it was effective in coconut fiber:peat potting mix. In no case was 1% biochar effective against powdery mildew. Yet, in all experiments, 3% biochar (of both kinds) was found to be effective against the studied foliar pathogens. Finally, as previously described for tomato and pepper plants (Elad et al. 2010), biochar in the potting medium granted long term protection against the various diseases.

Biochar amendment was found to induce the expression of the five studied defense-related genes, FaPR1, Faolp2, Fra a3, Falox and FaWRKY1, in non-infected strawberry plants, which is indicative of a direct effect of biochar to enhance strawberry plant resistance. FaPR1 belongs to the PR1 family, a classical marker for the SAR pathway. Faolp2, from the PR-5 family (Van Loon and Van Strien 1999), is one of two strawberry genes known to encode for thaumatin-like or osmotin-like proteins (OLP) (Wu et al. 2001; Zhang and Shih 2007). Acidic members of the PR-5 family, such as Faolp2, have been postulated to have anti-fungal properties (Zhao and Su 2010). Fra a3 is one of three known strawberry genes encoding for fruit allergens and belonging to the PR10 family (Munoz et al. 2010). PR10 proteins are small intracellular proteins which can exhibit antimicrobial activity in vitro against bacteria, fungi and viruses, and which are regulated by pathogen infection, various phytohormones, and abiotic stresses (Gomez-Gomez et al. 2011). Falox encodes a lipoxygenase, an enzyme which catalyzes the first step in transformation of linoleic and linolenic acids in oxylipins, and is involved in various aspects of plant development and response to biotic and abiotic stress (Porta and Rocha-Sosa 2002). Finally, FaWRKY1 codes for a protein with high sequence identity with WRKY75 from Arabidopsis (Encinas-Villarejo et al. 2009) and belongs to the WRKY trans-acting factors family involved in plant immunity (Pandey and Somssich 2009).

Although scant information is available on the actual involvement of these genes in the strawberry defense reaction, Faolp2 and FaWRKY1 expression were shown to be upregulated by SA, abscisic acid (ABA), wounding and upon infection by C. acutatum (Casado-Diaz et al. 2006; Encinas-Villarejo et al. 2009; Zhang and Shih 2007). In addition, we know from a previous study that FaPR1, Fra a3 and FaWRKY1 expression were induced upon drenching with an analogue of SA, Bion, and the biocontrol agent Trichoderma harzianum T39, while Falox was induced only by the application of T39, inducer of the ISR pathway (Meller Harel et al. 2011 ). This is suggestive that FaPR1 and Fra a3 are indicators of the SAR pathway, while Falox induction correlates with the ISR pathway of induced resistance. The simultaneous induction by biochar amendment of the SAR pathway (controlling the expression of FaPR1, Faolp2, and Fra a3) and the ISR pathway (controlling the expression of Falox) may explain how biochar amendment was able to affect the development of leaf fungal pathogens having different biological strategies of attack.

There is also some evidence that the presence of biochar induced a primed state against both B. cinerea and P. aphanis. Priming is defined as prior sensitizing of the plant immune system which enables an enhanced resistance to subsequent stresses, either biotic or abiotic (Conrath et al. 2006). Priming can be triggered by primary pathogen infection or by beneficial plant-microbe associations through induction of JA- and ET- responsive genes upon pathogen infection (Conrath et al. 2006; Van der Ent et al. 2009). Alternatively, certain chemicals can prime the plant for induction of SA-responsive genes, as in the case of protection against B. cinerea primed by ß-aminobutyric acid (BABA) in Arabidopsis (Zimmerli et al. 2001) or by hexanoic acid in tomato and Arabidopsis (Kravchuk et al. 2011; Vicedo et al. 2009).

In the studied systems, 1% GHW-450 biochar primed the plants for Faolp2 expression upon B. cinerea infection. Priming of expression of PR5 genes such as Faolp2 has been described in other systems, e.g., in Arabidopsis, ISR-priming by a nonpathogenic P. fluorescence treatment upon inoculation with P. syringae pv. tomato infection (Verhagen et al. 2004). Moreover, Falox expression appears to have been primed upon P. aphanis infection in plants grown on 3% biochar. Correspondingly, plants grown on 3% biochar were noticeably more resistant to anthracnose than plants grown on 1% biochar or control plants, according to the phenotypic AUDPC data. Therefore, it seems that in the case of the biotrophic disease, plant defense genes activation by biochar amendment is correlated with induced resistance.

The priming effect observed here appears to be similar to priming induced by PGPR as described previously by others (Pieterse et al. 2001; Verhagen et al. 2004). While soil microbial populations were not investigated in this work, studies have shown that biochar has a significant impact on microbial community structure in both the soil and the rhizosphere (Lehmann et al. 2011). Biochar was also reported to induce an increase in the relative abundances of bacterial phyla and genera with biocontrol capabilities (Kolton et al. 2011). In addition, a number of bacterial species isolated from biochar-amended potting medium and rhizosphere shared high sequence identity with strains known to have PGPR/F and biocontrol functions (Graber et al. 2010). Moreover, biochar contains a number of organic compounds belonging to various chemical classes (including n-alkanoic acids, hydroxy and acetoxy acids, benzoic acids, diols, triols, and phenols), some of which may directly elicit plant defense responses (Graber et al. 2010). For instance, various small diols and hydroxy acids (Graber et al. 2010) are similar in structure to bacterial organic compounds known to promote induced resistance via ethylene, jasmonic and salicylic acid pathways in Arabidopsis (Kwon et al. 2011). Whether the priming effect of biochar is a direct one stemming from biochar-borne chemicals, is elicited by biochar-induced PGPR and PGPF, or is the result of some combination of the two, is a critical question we are presently addressing. It is also not known how types of biochar, differing from one another in their chemical and physical characteristics, impact these and other plant responses in this and other plant-pathogen systems.

In conclusion, it is shown here for the first time that systemic resistance against foliar fungi adopting different infection strategies is induced in strawberry plants grown on biochar-amended medium. The molecular changes in defense-related gene expression which take place along both SAR and ISR pathways may contribute to the broad spectrum capacity of biochar for disease suppression.

Acknowledgements

The authors wish to thank Dr. Nir Dai and Dr. David Ezra for their help with the molecular work. This work was supported by grants from The Autonomous Province of Trento, Call for Proposal Major Projects 2006, Project ENVIROCHANGE and by the Chief Scientist of the Ministry of Agriculture and Rural Development of Israel, project number 301-0693-10. This paper is contribution no. 506/11 of the Agricultural Research Organization, The Volcani Center, Israel.

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© Springer Science+Business Media B.V. 2012