Biochar mediates systemic response of strawberry to foliar fungal pathogens
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.
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.
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.
The ability of biochar amendment to promote transcriptional changes along different plant defense pathways probably contributes to its broad spectrum capacity for disease suppression.
KeywordsBiotic stressInduced systemic resistancePlant diseasePrimingSystemic acquired resistanceSystemic resistance
real time quantitative PCR
induced systemic resistance
systemic acquired resistance
pathogenesis related proteins
plant growth-promoting rhizobacteria
plant growth-promoting fungi
biochar produced from citrus wood
biochar produced from greenhouse waste at 450°C
area under the disease progress curve
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
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.
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.
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
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.
Primers used in the qPCR experiments. Sequences are given in the 5′ to 3′ orientation
GenBank accession number or reference
Forward Primer (5′ to 3′)
Reverse Primer (5′ to 3′)
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
aca tgg gat gcc aat cta gc
cca cag gtt cac agc aga tg
aat tct ggg agc tgt caa cc
ggc aga aca caa ccc tat ag
ggg aga tgc tat cgg aga ca
ctt tcc ttt ccg gcc tta ac
agg caa atc ctc atc aat gc
gct cgg tga ata ccc agt gt
cat gca ttc tca tcc cat tg
acg gac agc agg cat atg ta
Effect of biochar on disease severity
Effect of CW and GHW-450 biochars on gray mold, anthracnose and powdery mildew
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).
Effect of plant age and exposure duration on induced resistance to gray mold by GHW-450 biochar amendment
Mature plants grown for 25 days in biochar
Daughter plants grown for 11 days in biochar
416B ± 50.5 aC
80 ± 17.0 a
157 ± 33.0 b
81 ± 18.6 a
110 ± 32.0 b
38 ± 9.6 b
Effect of growth medium on reduction of anthracnose on strawberry leaves by GHW-450 biochar amendment
207B ± 41.3 aC
159 ± 27.4 a
104 ± 18.2 b
165 ± 16.1 a
127 ± 14.2 b
81 ± 12.2 b
Expression of defense-related genes
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.
Combined pathogen and biochar effect on gene expression
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.
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.