Mechanisms governing the responses to anthracnose pathogen in Juglans spp.

Journal of Biotechnology 159 (2012) 251–264

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Mechanisms governing the responses to anthracnose pathogen in Juglans spp. P. Pollegioni a,∗ , G. Van der Linden b , A. Belisario c , M. Gras d , N. Anselmi e , I. Olimpieri a , L. Luongo c , A. Santini g , E. Turco g , G. Scarascia Mugnozza f , M.E. Malvolti a a

C.N.R. – Institute of Agro-environmental and Forest Biology, Viale Marconi 2, 05010 Porano, Terni, Italy Wageningen UR Plant Breeding, Wageningen University & Research Centre, PO Box 386, 6700 AJ Wageningen, The Netherlands C.R.A – Plant Pathology Research Center, Rome, Via C.G Bertero 22, 00156 Rome, Italy d C.R.A – Research Unit for Wood Production Outside Forests, Via Valle della Quistione 27, 00166 Rome, Italy e Tuscia University, Plant Protection Department, via San Camillo de Lellis, 0110 Viterbo, Italy f C.R.A. – Department of Agronomy, Forestry and Land Use, Via Nazionale 82, 00184 Rome, Italy g C.N.R. – Institute of Plant Protection, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy b c

a r t i c l e

i n f o

Article history: Received 26 January 2011 Received in revised form 28 July 2011 Accepted 8 August 2011 Available online 22 August 2011 Keywords: Walnut Host specific resistance Avoidance by late flushing NBS-profiling

a b s t r a c t Juglans nigra and Juglans regia are two highly economically important species for wood and fruit production that are susceptible to anthracnose caused by Gnomonia leptostyla. The identification of genotypes resistant to anthracnose could represent a valid alternative to agronomic and chemical management. In this study, we analyzed 72 walnut genotypes that showed a variety of resistance phenotypes in response to natural infection. According to the disease severity rating and microsatellite fingerprinting analysis, these genotypes were divided into three main groups: (40) J. nigra resistant, (1) J. nigra susceptible, and (31) J. regia susceptible. Data on leaf emergence rates and analysis of in vivo pathogenicity indicated that the incidence of anthracnose disease in the field might be partially conditioned by two key factors: the age and/or availability of susceptible leaves during the primary infection of fungus (avoidance by late flushing) and partial host resistance. NBS profiling approach, based on PCR amplification with an adapter primer for an adapter matching a restriction enzyme site and a degenerate primer targeting the conserved motifs present in the NBS domain of NBS-LRR genes, was applied. The results revealed the presence of a candidate marker that correlated to a reduction in anthracnose incidence in 72 walnut genotypes. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Anthracnose is caused by the fungus Gnomonia leptostyla (Fr.:Fr.) Ces. et De Not., anamorph Marssonina juglandis (Lib.) Magn. and is one of the most serious and widespread foliar disease of the Juglans genus (Belisario et al., 2008). Symptoms develop on the current season’s leaves, nuts, twigs and shoots as irregular necrotic areas that are often surrounded by small chlorotic halos (Belisario, 2002). Under favourable environmental conditions, these lesions may coalesce and cause fruit drop or poorly filled nuts, thus reducing the yield and quality of nut plantations (Woeste and Beineke, 2001; Belisario, 2002). In addition, repeated premature defoliation

∗ Corresponding author. Tel.: +39 0763374906; fax: +39 0763374980. E-mail addresses: [email protected] (P. Pollegioni), [email protected] (G. Van der Linden), [email protected] (A. Belisario), [email protected] (M. Gras), [email protected] (N. Anselmi), [email protected] (I. Olimpieri), [email protected] (L. Luongo), [email protected] (A. Santini), [email protected] (E. Turco), [email protected] (G. Scarascia Mugnozza), [email protected] (M.E. Malvolti). 0168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2011.08.020

in response to anthracnose may depress the annual growth of the stem diameter (Van Sambeek, 2003). Although several control methods have been applied to minimize the impact of G. leptostyla attacks, the selection of genotypes resistant to anthracnose disease could represent a valid alternative to both agronomic management (the application of nitrogen fertilizer, removing infected material) and chemical management (fungicides) (Berry, 1977). In Juglans nigra (black walnut) and Juglans regia (Persian walnut), complete resistance to the fungal agent of anthracnose has not yet been found, although a high differential response in susceptibility has been observed in the field (Funk et al., 1981; Woeste and Beineke, 2001; Anselmi et al., 2006). Persian and black walnut trees, which are able to efficiently circumscribe and overcome anthracnose infection, were classified as resistant genotypes. No single factor is responsible for the expression of resistance in the field, rather it is the result of the cumulative action of heredity, physiology and environmental factors (Calonnec et al., 2008). Nevertheless, Reid (1990) strongly suggested considering the putative relationship between the incidence of anthracnose in the field and the flushing progress of walnut trees during the vegetative season. As with other foliar diseases (Razdan and Gupta, 2009), anthracnose infection may be less severe when there is no

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synchrony between leaf maturation and time of primary infection by the pathogen (disease avoidance). According to Burdon (1987), disease avoidance is under the genetic control of the host and for this reason it is classified as a passive resistance mechanism. Numerous studies have shown that several classes of genes are involved in the timing of bud burst in forest tree species such as Quercus petraea (Derory et al., 2006), Castanea sativa (Casasoli et al., 2004; Santamaria et al., 2009) and Norway spruce (Yakovlev et al., 2006). They observed that bud burst is also mainly driven by temperature. In J. regia, the narrow-sense heritability estimates (Hanche et al., 1972) of bud-break date are extremely high (>90%). In addition, the significant north-south trend for mean site budbreak index among European provenances suggested the local adaptation to the climate conditions (Fady et al., 2003). As reported by Beineke and Masters (1973), adjacent J. nigra trees in plantations in North America frequently exhibit different levels of susceptibility to anthracnose. Genotypes derived from the western edge of the natural range of black walnut (Kansa and Oklahoma) are generally more susceptible. In addition, the resistance of black walnut trees in the field appeared to be highly heritable (∼70%). Mielke and Ostryl (2004) concluded that this differential disease response depends mostly on low selective pressure for anthracnose resistance in the above mentioned relatively arid regions. Alternatively, avoidance by late flushing is another plausible explanation: black walnut plants adapted to semi-arid steppe climates typical of Kansas and Oklahoma should flush early, implying a putative increased synchronization between leaf maturation and primary infection of G. leptostyla. Jacobs and Danielson (2002) detected a slight association between time of bud break and leaf emergence and responses of Fraxinus species in the field to anthracnose caused by the fungus Gnomoniella fraxini Redlin & Stack. Two late flushing ash species, the North American native F. quadrangulata and F. tomentosa, showed a lower probability of becoming infected, whereas the early budbreak Chinese ash F. chinensis was susceptible. These observations led us to verify whether the resistance frequently detected in walnut plantations is due to the mechanism of disease escape defined as avoidance by late flushing. Another aim was to verify if besides flushing differences among trees, host specific resistance plays a role in the occurrence of G. leptostyla infection. Host specific resistance to pathogens in plants is generally based on a gene-for-gene model: a host R-gene product recognises a specific pathogen gene product (effector), conferring full or partial resistance (Flor, 1971). Over the past decade, an increasing number of plant disease resistance genes (R-gene) have been cloned and characterized in mono- and dicotyledonous plants. Based on sequence comparisons and the conservation of structural features, these genes have been divided into four main classes (Martin et al., 2003). The most abundant class consists of receptor-like R-genes containing a nucleotide binding site-leucine rich repeat (NBS-LRR) structure. The NBS domain of the characterized R genes shows several highly conserved motifs including P-loop and kinase-2, which also occur in other proteins with ATP- and GTP-binding activities such as the apoptosis regulators Apaf-1 from humans and CED-4 from nematodes (Saraste et al., 1990; Meyers et al., 1999; Fig. 1). NBS-LRR proteins seem to be involved in recognizing pathogens and activating signal transduction pathways to induce plant defences (Hammond-Kosack and Jones, 1997). On invasion by biotrophic or specialized pathogens (pathogens adapted to overcome the specific defence mechanisms of plants), specific pathogen recognition governed by receptor-like R gene products usually leads to a hypersensitive response (HR). This takes place alongside the coordinated induction of an integrated set of defence responses, such as cell wall rigidification by the deposition of papillae and lignin, the accumulation of phenolic compounds, the synthesis de novo of phytoalexins, the extracellular generation of reactive

oxygen species, and the accumulation of pathogenesis-related proteins (Thordal-Christensen, 2003; Boller and He, 2009). As described by Mahoney et al. (2000) and Solar et al. (2006), juglone (5-hydroxy-1,4-naphoquinone), a phenolic compound endogenous to members of the Juglandaceae, may act as a protective agent against micro-organism infections. High levels of juglone in ontogenetically immature leaves have been correlated with a partial resistance to anthracnose in black walnut (Cline and Neely, 1984). A comparison between infected and uninfected leaves also proved that the host–pathogen interaction resulted in a disruption of juglone biosynthesis from its precursor hydrojuglone glucoside, catalysed by the HJG b-glucosidase enzyme (Duroux et al., 1998). At the beginning of G. leptostyla infection, a progressive decrement in juglone content may ensure rapid fungal development and pathogenic success. The detailed analysis of conidia germination, penetration and lesion development in the black walnut – G. leptostyla association revealed that host–cell reaction also involved the accumulation of granular cytoplasmic aggregates and the deposition of callose-containing papillae; when papillae were formed, no further penetration of the fungus was observed (Cline and Neely, 1983). Therefore, although the mechanisms underlying these results are still poorly understood, we postulated that besides late leaf-out mechanism, walnut resistance to anthracnose observed in the field may also be mediated by an interaction between an R-gene protein and an avirulent pathogen product. A novel molecular technique called nucleotide binding site (NBS) profiling has been developed in order to amplify a large collection of R-gene and R-gene Analogue (RGA) fragments and sample polymorphism in these genes (Van der Linden et al., 2004). NBSprofiling is a variant of the Motif-direct profiling approach (Van Tienderen et al., 2002) based on PCR amplification. It simultaneously uses an adapter primer that matches a restriction enzyme site and a degenerate primer by targeting the motifs highly conserved in the NBS domain of the NBS-LRR class of genes. The degenerate NBS primers were designed to anneal to as many NBS-LRR gene family members as possible while still being specific and repeatable. This method has been successfully used in apple (Calenge et al., 2005), potato (Malosetti et al., 2007; Wang et al., 2008; Brugmans et al., 2008), lettuce, barley, tomato (Van der Linden et al., 2004), and wheat (Mantovani et al., 2006), generating reproducible multilocus banding patterns that are highly enriched with RGA derived fragments. Over the last six years, the Institute of Agro-environmental and Forest Biology (Italian National Research Council, Porano, Italy) has been intensively involved in evaluating walnut germplasm in Italy. In collaboration with the Plant Protection Department of the University of Tuscia (Viterbo) and the CRA – Research Unit for Wood Production Outside Forests (Rome), a walnut plantation was selected and the resistance of J. nigra and J. regia genotypes to anthracnose in the field was analyzed (Annunziati et al., 2007). In this study, we present a detailed analysis of the reactions of these Juglans genotypes aimed at (1) verifying the correlation between leaf emergence and incidence of anthracnose in the field, and (2) detecting functional molecular markers that are tightly linked to R-gene and RGA (NBS-profiling) fragments correlated to resistance/susceptibility to anthracnose in walnuts.

2. Materials and methods 2.1. Plant material 2.1.1. Experimental plantation This study was carried out in a 21-year-old plantation made up of many Juglans species located in the experimental farm of the CRA – Research Unit for Wood Production Outside Forests (Azienda

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Fig. 1. The targeted cytoplasmic nucleotide-binding site – leucine-rich repeat (NBS-LRR) – containing R-gene sequences. Shaded boxes indicate the highly conserved motifs (P-loop, kinase2, GLPL) in the NBS domain region. Relative position and orientation (horizontal arrows) of primers that target conserved motifs are shown (NBS1, NBS5a6).

Ovile, Borgata Casalotti, Rome, Italy, latitude 41◦ 54 56 N, longitude 12◦ 22 9 E, 40 m a.m.s.l.). This experimental plantation was established in 1989 in order to evaluate the growth performance of J. regia, J. nigra, J. × intermedia Carr. (NG23 × RA) hybrids and J. major × J. regia hybrids (MJ209 × RA). Randomly seeds of J. regia, and J. nigra (Italian germplasm) were provided by the Italian Società Agricole e Forestale (SAF) nurseries of Sorrento (Naples) and Casale Monferrato (Alessandria), while the hybrids were provided by the French INRA nursery of Orleans (Dr. E. Germain).

from the open-pollination of JN044 tree (two year-old half sib family) with only one scion on each rootstock. Each rootstock was carefully checked by microsatellite marker analysis (Malvolti M.E. and Gras M., data unpublished). The success of the vegetative propagation was lower than expected and for each genotype only three out of ten clones were obtained. They were placed in a greenhouse and used for in vivo pathogenicity tests.

2.1.2. Walnut genotypes with differential responses to anthracnose in the field In summer 2004, a detailed study of the plant response to anthracnose disease in the field was carried out on a subset of 72 different genotypes which were randomly selected from the Azienda Ovile plantation. According to a preliminary morphological observation of the leaf traits, 41 trees were classified as J. nigra and 31 as J. regia (Table 1). Plant response to anthracnose attack by natural infection was evaluated in the field at the beginning of August, when the secondary infections by M. juglandis conidia dissemination were expected to occur and necrotic symptoms were clearly visible. Three compound leaves per tree were collected at three heights (3, 5, 7 m) of the canopy from 7 to 10 August 2004. As reported in Table 1, the mean number of necrotic spots (small, medium, large) per leaflet and the percentage of necrotic leaflet area were recorded for each genotype. According to the relative differences in disease severity rating (DSR), this subset of 72 genotypes was further divided into two main groups: 40 J. nigra resistant, and 32 (31 J. regia and 1 J. nigra) susceptible plants in the field (Annunziati et al., 2007). Subsequently, the former classification of 72 walnut genotypes was confirmed by applying the efficient and quick method proposed by Woeste and Beineke (2001). According to this approach, from 7 to 10 August 2008, DSR in the field were assigned on a scale from 1 (=no or a few lesions) to 5 (=leaves completely senescent or abscised), based on the appearance of the entire tree (Table 1; data kindly provided by CRA – Research Unit for Wood Production Outside Forests of Rome). During early spring 2008, young and healthy leaves were sampled from each selected walnut tree for genomic DNA extraction and stored after shock-freezing at −80 ◦ C until use. At the end of February 2008, ten scions were collected from each tree and used for clonal propagation. In the Umbria regional forestry nursery (UmbraFlor s.r.l., Spello, Perugia, Italy), the 72 walnut genotypes under investigation were propagated by grafting according to the “hot-callusing cable” approach successfully applied in Juglans genus by Siniscalco (1994a,b). Ten scions collected from each Persian walnut genotype (RA004–RA128) were grafted onto ten cv. “Sorrento” rootstocks, with only one scion on each rootstock. Similarly ten scions collected from each black walnut genotype (JN001–JN126) were grafted onto ten J. nigra rootstocks obtained

In summer 2004, plant response to anthracnose in the field was evaluated, however the flushing progress of walnut trees was observed but not recorded by Annunziati et al. (2007). Therefore, from April to June 2008, the development stage for bud/shoot for the 72 walnut trees in the Azienda Ovile plantation was recorded weekly, following a modified approach of Hemery et al. (2005). Flushing was scored on an eight-point scale: stage 0 = bud closed; stage 1 = bud breaking; stage 2 = bud breaking with visible leaves; stage 3 = emerging leaves < 2 cm long; stage 4 = emerging leaves > 2 cm long; stage 5 = expanding shoot < 4 cm growth; stage 6 = expanding shoot > 4 cm growth; stage 7 = full expanded leaves (Fig. 2). When more than 75% of the canopy exhibited buds/shoots at one of the eight possible stages, the corresponding point scale was assigned. Progression of the leaf emergence rate was recorded in Julian days (Jd) from 1 January 2008. The first annotation was made on 1 April 2008 (Jd 79) and repeated every seven days, until 8 June 2008 (Jd 149). For each tree the flushing data were summarized in terms of the number of Julian days required to reach the full expanded leaf stage 7.

2.2. Leaf emergence rate analysis

2.3. In vivo pathogenicity tests The strain ISPaVe2000 of G. leptostyla (CRA-PAV3033; http://www.colmia.it/bancaDati.cfm) was used to screen the susceptibility of the 72 walnut genotypes. Two 2-year old clones per genotype were inoculated by applying the procedure described by Belisario et al. (2008). Under greenhouse conditions the potted clones were sprayed to run off with a suspension of 1 × 105 conidia ml−1 when leaf blades were fully expanded in both Juglans species (June 1st 2010). Inoculum was prepared by suspending conidia in sterile water; conidia were picked from conidiomata produced in 1-month-old colonies grown on PDA at 22 ◦ C in the dark. After being sprayed, clones were covered with transparent polythene bags for 48 h. The test was conducted in controlled conditions at approximately 25 ◦ C, relative humidity 60%, and under natural light. Control plants (one clone per genotype) were sprayed with sterile water and kept under the same conditions but in a separate block to avoid cross contamination. When they had been removed from the plastic bag, the clones were arranged into two blocks. Twenty one days after inoculation,

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Table 1 Walnut anthracnose disease severity rating for 41 J. nigra and 31 J. regia 21-year old trees assessed as a mean number of necrotic spots (small, medium, large, total) per leaflet and percentage of necrotic area per leaflet (Annunziati et al., 2007) during summer 2004 and using ) scoring system during summer 2008. Walnut genotypes showing susceptibility in both surveys (disease rating >1) are reported in bold. Disease severity rating (DSR) observed in the experimental field Mean number of necrotic Necrotic leaflet J. nigraa spots per leafletd area IDb

Smallc

JN001 JN005 JN013 JN014 JN018 JN020 JN032 JN044 JN051 JN057 JN058 JN060 JN061 JN062 JN068 JN069 JN070 JN071 JN074 JN076 JN078 JN082 JN083 JN087 JN088 JN089 JN090 JN095 JN096 JN100 JN101 JN102 JN111 JN113 JN114 JN115 JN116 JN117 JN118 JN123 JN126

11.2 18.7 6.9 10.2 21 23.2 19 21 9.8 7.5 13.5 14.9 9.8 16.4 12.1 8.7 9.0 6.7 16 11.5 16 18.3 5.8 35 11.2 12.8 18.5 15 17.2 12.3 8.2 16 11 5.8 14.8 13.9 6.4 11.4 9.2 15.7 10.8

a b c d e

Mediumc

0.2

Largec

Scoree

(%) 0.08 0.07 0.02 0.03 0.10 0.10 0.08 0.10 0.02 0.02 0.05 0.04 0.05 0.05 0.04 0.05 0.06 0.04 0.07 0.06 0.07 0.10 0.04 1.50 0.03 0.04 0.08 0.07 0.07 0.05 0.04 0.06 0.03 0.04 0.07 0.04 0.02 0.03 0.03 0.07 0.10

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

J. regiaa

Mean number of necrotic spots per leafletd

IDb

Smallc

Mediumc

Largec

(%)

RA004 RA012 RA015 RA029 RA033 RA034 RA035 RA036 RA037 RA038 RA039 RA041 RA042 RA045 RA047 RA048 RA049 RA053 RA054 RA055 RA064 RA073 RA077 RA081 RA094 RA103 RA108 RA110 RA121 RA122 RA128

70 3713 811 1868 839 559 778

0.8 500 1.1

0.1 0.85 0.2

5.4 42.3 9 33 7.7 5 7.2 3.3 12 42.5 4.8 9.8 10.2 7.5 6.3 8.1 6.7 7.7 12 7.4 33 37 5.3 21 4 6 8 8.2 6 5 1.3

Necrotic leaflet area

1 2 23

733.3 2425 32.5 514 653 797.5 485 545.2 259 289 431 830 839

45800 549

250

0.6 9.31 1 364 25.7 6.7

1.7

0.2 3.5

8.7

41.3 64.6 427

11.6 53.4 29.5 5

2.9

Scoree

3 5 3 4 3 3 3 3 3 5 3 3 4 3 3 3 3 3 3 3 4 4 3 4 3 3 3 3 3 3 2

The preliminary classifications of black and Persian walnut trees were based on morphological observations of leaf traits. ID = genotype identity. Diameter of anthracnose necrotic spots: large >5 mm; medium 1–5 mm; small <1 mm. A total of 42 and 27 leaflets were evaluated in each J. nigra and J. regia genotype, respectively. Woeste and Beineke (2001) scoring system.

two compound leaves were collected at two levels (base, top) per clone and the disease severity rating (DSR) of each clone was determined by computing the percentage of leaflets with one or more anthracnose spots, the mean number of small (diameter of spots <1 mm), medium (1–5 mm) and large (>5 mm) necrotic spots per leaflet. The mean percentage of necrotic area per leaflet was also considered. 2.4. Genomic DNA extraction For each sample of the 72 genotypes tested, 100 mg of leaf tissue was homogenized in a 2-ml microcentrifuge tube containing a 5 mm steel bead cooled with liquid nitrogen using Mixer Mill 300 (Qiagen, Hilden, Germany). Genomic DNA was extracted and purified using the DNeasy96 Plant Kit (Qiagen) according to the manufacturer’s instructions (http://www.qiagen.com), and stored at −20 ◦ C. The quantity and quality of genomic DNA was assessed using a NanoDrop ND – 1000 spectrophotometer

(NanoDrop, Wilmington, DE, USA). The DNA in the samples was brought to a working concentration of 5 ng/␮l for microsatellite analysis and 20 ng/␮l for NBS analysis. 2.5. Microsatellite analysis In order to confirm the identity of the 72 walnut genotypes and exclude the presence of putative hybrids in our collection, a fingerprinting analysis was carried out using microsatellite (SSR) markers. Ten unlinked microsatellite loci (WGA1, WGA4, WGA9, WGA69, WGA89, WGA118, WGA202, WGA276, WGA321, WGA331) already selected, sequenced and used for the retrospective identification of hybridogenic plants in Juglans spp. and for genetic characterization of J. × intermedia trees (Pollegioni et al., 2009) were amplified in all samples. Both the expected allelic range and the expected private and common alleles between J. nigra and J. regia species for each SSR locus are reported in Table 2. The PCR amplification and the visualization of amplified SSR alleles for each

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Table 2 List of ten nuclear microsatellites already sequenced and used for retrospective identification of hybridogenic plants in Juglans spp. and for characterization of J. × intermedia trees (Pollegioni et al., 2009). For each SSR, the expected allelic range (bp), expected species-private alleles (bp), expected common alleles (bp), total number of alleles amplified on the 72 walnut plants collected in the experimental plantation of Azienda Ovile site and new amplified alleles are shown. SSR locus

Expected allelic range (bp)

WGA1

179–211

WGA4

231–257

WGA9

228–261

WGA69 WGA89

159–179 186–234

WGA118

183–244

WGA202

246–295

WGA276

144–195

WGA321

226–195

WGA331

177–274

Total

Expected species-private alleles (bp)

Expected common alleles (bp)

Total number of amplified alleles

New amplified alleles (bp)

10

179

J. nigra

J. regia

181–183–185–187–196 198–200–211 240–242–246–248–250 252–257 228–241–251–255–257 261 169–171–173–177 190–196–201–203–207 213–219–222–226–230 234 210–212–214–221–223 226–236–244

180–188–192–194

189–190

231–235

233–237

10

254

239

243–247

9

253

159–161–167 215–221

175–179 211

7 13

– 186

183–196–198–206

–

14

208–219

265–267–275–295

260

10

228–242 –

171–173–175–177–179 181–183–187–189–191 195 226–230–239–241–243 245 270–274

–

16

–

–

13

–

10

238–252 260 199

246–248–250–252–254 256–258 144–147–149–153–155 157–159–161–163–165–167–169 236–237–242–244–246 248–254–264 177–179–181–183–185 187–189–191–195

112

Fig. 2. Flushing scoring system used to assess leaf emergence rate in the black and Persian walnut trees according to the modified approach of Hemery et al. (2005). (A) 0 – bud closed; (B) 1 – bud breaking; 2 – bud breaking: visible leaves; (C) 3 – emerging leaves: <2 cm long; (D) 4 – emerging leaves: >2 cm long; (E) 5 – expanding shoot: <4 cm growth; (F) 6 – expanding shoot: >4 cm growth; (G) 7 – full expanded leaf.

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sample were carried out as described by Pollegioni et al. (2009). The amplified SSR fragment data were collected using Gene Scan Analysis version 3.7 Software and genotype profiles were assigned with the Genotyper version 3.7 NT Software (Applied Biosystems, Foster City, CA, USA) using three J. regia and three J. nigra genotypes already characterized by 10 SSR markers as a standard across multiple plates. 2.6. NBS profiling procedure Nucleotide binding site profiling of walnut genotypes was carried out as described by Van der Linden et al. (2004), with some modifications. The restriction digestion and adaptor ligation events were combined in one single reaction of 60 ␮l, consisting of 200 ng of genomic DNA, 1 mM ATP, 10 U restriction enzyme, 1 U T4 ligase for sticky enzyme (5 U for blunt enzyme) and a restriction/ligation buffer (10 mM Tris·HAc pH 7.5, 10 mM MgAc, 50 mM KAc, 5 mM DTT, 50 ng/␮l BSA). After incubation for 3 h at 37 ◦ C, the reaction was stopped by heat inactivation (15 min at 65 ◦ C). The restriction/ligation mixture was diluted twice and used as a template for PCR. NBS-specific fragments were amplified in a single polymerase chain reaction with an NBS-primer and adapter primer rather than the two-step PCR procedure in the original protocol. PCR was done in 25 ␮l of reaction volume by adding 5 ␮l of restriction-ligation template, 2.5 ␮l of HotStartTaq PCR buffer 10×, 200 ␮M dNTPs, 20 pmol for each primer and 0.4 U of HotStartTaq polymerase (Qiagen). Reactions were performed in a GeneAmp 9700 Thermocycler according to the following procedure: 15 min at 95 ◦ C (to activate HotStartTaq polymerase) followed by 30 cycles of 30 s at 95 ◦ C, 1.40 min at 55 ◦ C, and 2 min at 72 ◦ C; with a final extension step at 72 ◦ C for 20 min. The amount and estimated size of the amplified fragment was checked testing 15 ␮l aliquot of the amplified reaction by electrophoresis on 1% agarose gel in 0.5× TBE buffer, and stained with ethidium bromide. Finally, the labelling PCR was performed as described by Brugmans et al. (2008). The NBS primers were used in combination with a non-selective adapter primer labelled with the near-infrared fluorescent dye IRD700. PCR was done in 10 ␮l of reaction volume by adding 5 ␮l of 10× diluted PCR mixture, 1 ␮l of PCR buffer 10×, 200 ␮M dNTPs, 3 pmol of NBS primer, 0.6 pmol of IRD700-labelled adapter primer and 0.2 U of SuperTaq DNA polymerase (Qiagen). Reactions were performed according to the following procedure: 3 min at 95 ◦ C followed by 35 cycles of 30 s at 95 ◦ C, 1.40 min at 55 ◦ C, and 2 min at 72 ◦ C; then a final extension step at 72 ◦ C for 20 min. The labelled PCR products were mixed with an equal volume (10 ␮l) of formamide-loading buffer (98% formamide, 10 mM EDTA pH 8.0 and 0.1% Bromo Phenol Blue) and an aliquot (0.3 ␮l) was denatured at 95 ◦ C for 5 min, and immediately chilled on ice. Amplification fragments were visualized on a denaturing polyacrylamide gel using an NEN® IR2 DNA analyser (Li-COR® Biosciences, Lincoln, NE, USA). Each gel contained four duplicate samples to evaluate the reproducibility of the protocol. In this study, two different degenerate NBS primers, NBS1 and NBS5a6 (NBS5a combined with NBS6 in 1:1) and two restriction enzymes, MseI and RsaI, were used for a total of four NBS primer–enzyme combinations. The positions of the NBS1 (5 -GCIARWGTWGTYTTICCYRAICC-3 ) and NBS5a6 (NBS5a: 5 -YYTKRTHGTMITKGATGAYGTITGG-3 ; NBS6: 5 YYTKRTHGTMITKGATGATATITGG-3 ) primers in the NBS domain are shown in Fig. 1. These primers were designed from part of the conserved P-loop motif for NBS1 and of kinase-2 for NBS5a6; NBS1 primer amplified DNA towards the 5 end of the targeted genes, outside the NBS region, meanwhile NBS5a6 towards the 3 end, inside the NBS region. For each enzyme–NBS primer combination, only repeatable bands were considered: ambiguous bands were

either recorded as missing data or excluded from the analysis. The NBS bands were scored on the basis of their presence (1) or absence (0), and summarized in a binary data matrix. 2.7. Sequence analysis of candidate NBS fragment Candidate NBS fragment was excised from LI-COR gels as described in the Odyssey® manual for “AFLP band cut out and band extraction” (Westburg, The Netherlands) and re-amplified with conditions identical to the first PCR of the NBS profiling protocol. The amplified fragment were checked on agarose gel, purified with a QIAquick PCR purification Kit (Qiagen) and cloned into the pGEMT Easy Vector (Promega, Madison, WI, USA) prior to sequencing with M13 forward or M13 reverse primers. The sequencing reaction was carried out in a total volume of 10 ␮l containing 0.5 ␮l of template DNA (PCR product), 1 ␮l of 5× BigDye Terminator v. 1.1 Buffer, 3 ␮l of BigDye Terminator v. 1.1 Ready Reaction Mastermix (Applied Biosystem), and 2.5 ␮l primer (2 pmol/␮l). The sequencing thermal profile was 1 min at 94 ◦ C, followed by 25 cycles of 94 ◦ C for 20 s, 50 ◦ C for 15 s, and 60 ◦ C for 1 min. Sequencing reaction products were purified by SpinColumns kits (Princeton/Applied Biosystem) and run on the ABI PRISM 3700 Genetic Analyzer. Sequences were analyzed and edited with the Sequence analyzing software 3.7 (Applied Biosystem) and compared to the public non-redundant (nr) Viridiplantae database (GenBank database http//www.ncbi.nlm.nih.gov) using BLASTN. All BLAST hits with expect value (E) > 1.0E−05 were eliminated. 2.8. Data analysis A Mann–Whitney test (equivalent non-parametric t-test; Hollander and Wolfe, 1999) was conducted to detect a statistical difference in the leaf emergence rate between J. regia and J. nigra tree groups selected in the experimental plantation of Azienda Ovile. The putative correlation between the disease severity rating (DSR) versus anthracnose observed in the field and the flushing progress of the 31 J. regia and the 41 J. nigra genotypes was explored. The Spearman rank correlation between the number of Julian days required to reach the full expanded leaf stage 7 per tree (season 2008), the mean percentage of necrotic leaflet area (season 2004), and the disease rating according to the Woeste and Beineke (2001) method (season 2008) were calculated. Lack of phenological data prevented us from computing the Spearman coefficient between leaf-out Julian days during season 2004 and the anthracnose disease incidence in the field. The Spearman rank correlation between the percentage of spotted leaflets, mean number of necrotic spots per leaflet (small, medium, large) and mean percentage of necrotic area per leaflet measured for each genotype by in vivo pathogenicity tests were also calculated. The rank-based Kruskall–Wallis test (alternative non-parametric ANOVA-test; Hollander and Wolfe, 1999) was used to determine walnut genotype differences in relation to anthracnose susceptibility measured in vivo by inoculation with the ISPaVe2000 strain of G. leptostyla. In addition, pairwise genotype comparisons for in vivo disease incidence of walnut plants were performed based on a non-parametric post hoc Dunn test (Dunn, 1964). The corresponding P values of Kruskall–Wallis, Dunn and Mann–Whitney tests were not computed asymptotically but by adopting a permutation approach with 105 Monte Carlo re-samplings. All computations were performed by XLSTAT2010 software (http://www.xlstat.com). The genetic classification of the 72 walnut trees (genotypes) selected in the experimental plantation of Azienda Ovile was definitively performed by assigning the genotypes to one of the three classes (J. nigra, J. regia and J. × intermedia) using SSR markers. Two assignment tests were conducted using GENECLASS 2

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software (http://montpellier.inra.fr/CBGA/softwares/): the Paetkau et al. (1995) frequency method, and Rannala and Mountain’s (1997) partial Bayesian method. Both approaches removed the individual being assigned (leave one out procedure), computed the allelic frequencies in all candidate classes (assuming the HWE), calculated the likelihood of the individual’s multilocus genotypes occurring in each class (independence of loci), and assigned the individual to the class with the highest likelihood. Missing alleles were assigned an arbitrary nonzero frequency (0.01). In order to evaluate the genetic relationships between genotypes, two distinct UPGMA (Unweighted Pair Group Method with Arithmetic mean) trees were constructed based on SSR and NBS markers. The Simple Match coefficient (SM – Sokal and Sneath, 1963) for dominant functional NBS markers and the Band-Sharing coefficient (Lynch, 1990) for co-dominant neutral SSR markers were calculated between all pairwise combinations of individuals. The statistical correlation (r) between SM and Band-Sharing similarity matrices was calculated by Mantel’s non-parametric multivariate test (Mantel, 1967) with 1000 permutations. All computations were carried out with NTSYSpc version 2.1 software package (Rohlf, 2001). Bootstrap support for UPGMA trees was determined by re-sampling loci 1000 times using WinBoot software (Yap and Nelson, 1996). Descriptive gene diversity statistics, the mean effective number of SSR alleles or NBS bands (Ne ), Nei’s (1973) gene diversity index (He ), Shannon’s diversity index (I), and percentage of polymorphism (P) were estimated for SSR and NBS markers with GenAlEx version 6 software (Peakall and Smouse, 2005) and POPGENE version 1.32 programme (Yeh et al., 1997; http://www.ualberta.ca/∼fyeh/index.htm) respectively. Shannon’s diversity index is a useful tool for dominant data analysis, due to its relative insensibility to the bias produced by failures to detect heterozygous individuals and the advantage of not assuming the Hardy–Weinberg equilibrium. In addition, in order to explore the power of NBS and SSR markers to identify individuals, we also computed the Power of Discrimination (PD) coefficient according to Kloosterman et al. (1993). A PD coefficient provides an estimate of the probability that two randomly sampled genotypes would be differentiated by their allelic profiles. Two different measures of genetic differentiation among species were calculated via AMOVA (Excoffier et al., 1992) implemented in Arlequin software version 3.11 (Excoffier et al., 2005) using SSR markers: Fst coefficient (Weir and Cockerham, 1984), based on the Infinite Alleles Mutation Model of loci (IAM), and Slatkin’s (1995) Rst coefficient which takes into account a stepwise mutation model (SMM). The analogous Fst coefficient for binary NBS data (population ˚PT value; Excoffier et al., 1992) was also computed via AMOVA. The statistical significance of Fst , Rst and ˚PT was tested using a non-parametric approach described in Excoffier et al. (1992) with 1000 permutations. In addition, we used SMOGD 1.2.5 software (Crawford, 2009) to measure the actual differentiation coefficient (Dest ) overall SSR loci among species according to Jost (2008).

3. Results 3.1. Leaf emergence notations Extensive variability in emergence stages was noted in 41 J. nigra and 31 J. regia trees in the experimental plantation. On 1 April 2008 (Jd 79) Persian walnut tended to be earlier to leaf out with over 74% of the trees at stage 4 (emerging leaves > 2 cm long) and 5 (expanding shoot < 4 cm growth). All mean leaf emergence score values of the J. regia group were higher than those measured in the J. nigra group (Fig. 3A). The average difference in leaf development

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Fig. 3. (A) Mean leaf emergence phenological stage of 41 J. nigra (䊉) and 31 J. regia () trees at Azienda Ovile, CRA – Research Unit for Wood Production Outside Forests (Rome) in spring 2008 versus Julian days (days from January 1st). (B) Comparison between ( ) the mean number of days required to reach the full expanded leaf stage (Jd ± SE) and ( ) the mean incidence of anthracnose lesions in the field (percentage of necrotic leaf area ± SE) for J. nigra and J. regia tree group measured in summer 2004.

between black and Persian walnut trees ranged from 3.870 to 0.097 points measured on the 1 April 2008 (Jd 79) and 26 May 2008 (Jd 135) respectively. Thus no difference in flushing progress score was computed after 2 June 2008 (Jd 142) when fully expanded leaves were found in 100% of the 72 walnut trees. In the second half of April (Jd 93), when the primary infection of G. leptostyla is supposed to start and ascospores are released (Belisario et al., 2008), the mean emergence score of J. regia species was 5.129 ± 0.562 with 67.74% of the trees showing shoots expanding (stage 5) and 22.5% had shoots longer than 4 cm. On the other hand the mean flushing score of J. nigra species was 1.03 ± 1.03 (Fig. 3A). By the end of April about 25% of black walnut trees had completely closed buds and the remaining 31 plants (75.6%) did not exceed stage 3 (emerging leaves < 2 cm). No considerable variation in flushing score was observed within each species. According to Mann–Whitney’s non-parametric test (P < 0.001), the mean number of Julian days required to reach leaf stage 7 (full expanded leaf) were statistically different in J. nigra (131.24 ± 4.97) and J. regia (108.13 ± 2.61) genotypes (Fig. 3B). The Spearman rank analysis for non-parametric data detected a significant negative correlation (r = −0.791, P < 0.01) between the mean number of Julian days required to reach leaf stage 7 and the incidence of anthracnose in the field assessed as a mean percentage of the necrotic area of leaflets in summer 2004. As reported in Table 1, 40 J. nigra genotypes showed only a few small necrotic spots per leaflet and an extremely low percentage of necrotic areas per leaflet (≤0.1). Conversely, one J. nigra (JN087) and 31 J. regia genotypes presented a relatively high percentage of leaflet necrotic areas (≥1.25). In summer 2008 the relationship between flushing progress per tree and anthracnose effect quantified using Woeste and Beineke’s disease severity scoring system (2001) was also highly significant (r = −0.924, P < 0.01).

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3.2. In vivo pathogenicity test An unequivocal differentiation between resistant and susceptible genotypes to anthracnose in the 72 walnut plants was achieved by inoculating the mature, fully expanded leaves of each tree with the strain ISPaVe2000 of G. leptostyla. Twenty one days after the inoculation, the percentage of leaflets with one or more anthracnose spots, the mean number of small (diameter of spots < 1 mm), medium (1–5 mm) and large (>5 mm) necrotic spots per leaflet and the mean percentage of necrotic leaf area per leaflet were computed for each Persian and black walnut genotype (Supplementary Table 1). As expected, no significant block effects or interaction between blocks were found and no necrotic lesions were detected in the uninoculated plants (control plants). Nevertheless, the rank-based Kruskall–Wallis test revealed statistically significant differences in the in vivo-disease severity rating (DSR) among the 72 walnut trees (P < 0.0001). As shown in Fig. 4, the post-hoc paired comparison analysis for non-parametric data by Dunn’s test divided the inoculated samples into two major groups (P < 0.05). The first group incorporated 40 J. nigra genotypes (JN001–JN126) previously classified as resistant to anthracnose in the field. Their in vivo-mean percentage of necrotic leaf area per leaflet ranged from 0.04 (JN001) to 0.52 (JN020) with an overall mean value of 0.255 ± 0.143 (Supplementary Table 1). The above mentioned 40 J. nigra plants were considered to be “partially resistant” to G. leptostyla attack. The second group, which included 31 J. regia trees (RA004–RA128) and one J. nigra tree (JN087) with relatively high susceptibility in the field (Table 1), was characterized by an in vivo-mean percentage of necrotic leaf area per leaflet values varying from 1.12 (RA037) to 4.87 (RA004), with an overall mean value of 2.001 ± 0.775 (Fig. 4). These 32 walnut genotypes were significantly less resistant to anthracnose than the 40 J. nigra genotypes and classified as “susceptible”. In addition, highly significant rank correlations were found between the mean percentage of the necrotic leaf area per leaflet and the percentage of spotted leaflets (r = 0.874, P < 0.001), the mean number of small spots (r = 0.774, P < 0.001), medium spots (r = 0.829, P < 0.001), and large spots (r = 0.879, P < 0.001) per genotype, thus confirming the previous classification (Supplementary Table 1). 3.3. SSR fingerprinting analysis The ten selected microsatellites gave amplification in all the 72 walnut plants and produced fragments of variables size (Table 2). The final classification of the 72 walnut trees was conducted by comparing their SSR profiles against 343 J. nigra, 49 J. regia and 205 J × intermedia genotypes that had already been characterized and identified by Pollegioni et al. (2009). The assignment tests, the Paetkau et al. (1995) frequency method and Rannala and Mountain (1997) partial Bayesian method assigned 41 genotypes (JN001–JN126) to the J. nigra group and the remaining 31 genotypes (RA004–RA128) to the J. regia group. This confirmed their preliminary classification based on the morphological observation of leaf traits, and ruled out the presence of putative J. × intermedia hybrids in our set of samples (Table 1). In addition, the UPGMA tree based on the Band-Sharing similarity coefficient provided additional insights into the relationships between walnut trees (Fig. 5B). As expected, two major clusters were detected with bootstrap values of 99%, which corresponded exactly to the two groups inferred via assignment analysis. This cluster-tree did not reveal any genetic sub-structure within each inferred cluster. However, the microsatellite loci showed a high level of variability in the tested samples. The main diversity parameters detected in 41 black walnut and 31 Persian walnut trees, including an effective number of SSR alleles (Ne ), Nei’s (1973) gene diversity index (He ), Shannon’s diversity index (I), the percentage of polymorphism (P)

are reported in Table 4. The probability that two randomly sampled genotypes would be differentiated by their SSR profiles was extremely high in each species. Across all loci the Power of Discrimination (PD) values ranged from 0.974 for J. nigra to 0.967 for J. regia (Table 4). The hierarchical locus-by-locus AMOVA analysis revealed that the majority of molecular variance (68%) resided within groups, while 32% were distributed among groups using the FST statistic (FST = 0.315). By contrast, the RST statistic partitioned 92% of the variation between groups and 8% within groups (RST = 0.928). In addition, the mean actual differentiation Dest coefficient (Jost, 2008), an alternative measure of genetic differentiation, was 0.871 overall SSR loci. 3.4. NBS profiling analysis In total four NBS-primer/enzyme combinations were tested on the 41 black walnut and 31 Persian walnut trees (Table 3). They produced a total of 317 reproducible fragments, ranging from 117 in MseI–NBS1 to 33 in RsaI–NBS5A6 combinations. Out of 317 NBS bands scored in this germplasm collection, 145 and 125 were private to J. nigra and J. regia, respectively. The remaining 47 NBS bands were shared between the walnut species and classified as common NBS bands (Table 3) although the homoplasy events cannot be ruled out. A high proportion of the private NBS fragments were monomorphic. The percentage of polymorphism (P) varied from 45.90% (MseI–NBS1) to 60.80% (MseI–NBS5A6) for J. nigra, and from 32.60% (MseI–NBS5A6) to 60.40% (MseI–NBS5A6) for J. regia with a mean value of 54.10% and 46.30%. Although J. nigra and J. regia displayed a moderate level of variability, the chance of finding two individuals with the same NBS profile in each group was almost null: the Power of Discrimination values ranged from 0.966 in black walnut to 0.943 in Persian walnut trees (Table 4). In order to evaluate the genetic relationships between 72 walnut genotypes, a UPGMA tree was constructed based on the Simple Match coefficient values calculated between all pairwise combinations of individuals using four NBS-primer/enzyme combinations. As highlighted in Fig. 5A, the NBS profiling dendrogram showed two main clusters supported by a robust bootstrap analysis (P = 100%, 1000 replicates). These two inferred clusters were obtained using neutral SSR markers (Fig. 3B), and included 31 J. regia genotypes (RA004–RA128) and 41 J. nigra genotypes (JN001–JN126). Genetic substructures within each walnut group appeared negligible with a bootstrap support lower than 70%. A significant and positive correlation was found between NBS and SSR individual pairwise genetic similarity matrices by Mantel’s test (r = 0.879, P ≤ 0.01). This positive correlation was largely due to the strong genetic differentiation between J. nigra and J. regia plants. AMOVA analysis based on 317 NBS bands confirmed that most of the diversity was located between species, attributing 86.96% of the molecular variance between groups and 13.64% within groups (FPT = 0.864, P < 0.01; Table 4). Finally, a detailed analysis of NBS marker distributions revealed the presence of a putative candidate NBS marker correlated to anthracnose susceptibility in walnut (Fig. 6). In the NBS1–RsaI combination, all in vivo-susceptible plants, 31 J. regia (RA004–RA128) and one J. nigra (JN087), showed a common band of 91 bp in length, which was not present in the 40 in vivo-partially resistant J. nigra plants (JN001–JN126). The candidate band amplified in JN087 susceptible J. nigra and three susceptible J. regia genotypes randomly chosen (RA004, RA041, RA029), was excised from polyacrylamide gel, re-amplified, cloned and sequenced. The DNA sequence analysis of colonies containing recombinant vectors revealed that this short band (91 bp) consisted of two distinct fragments, NBS1–RsaI91bpA (GeneBank accession no. HQ993069) and NBS1–RsaI-91bpB (GeneBank accession no. HQ993070) with identical sizes in both genotypes. These two sequences showed no significant homology

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Fig. 4. Disease severity rating (DSR) of 31 J. regia () and 41 J. nigra ( ) genotypes assessed as a mean percentage of necrotic leaf area calculated in the field during summer 2004 (dotted line; Annunziati et al., 2007) and in vivo by inoculation with strain ISPaVe2000 (bar). Mean values showing the same letter are not significantly different at P ≤ 0.05 according to the post hoc non-parametric Dunn test (Dunn, 1964).

Fig. 5. UPGMA trees of the 41 J. nigra and 31 J. regia genotypes based on 317 NBS functional markers (A) and 10 neutral SSR markers (B). Bootstrap P values based on 1000 replicates are reported at the corresponding node for each cluster. P values lower than 20% are not shown. * indicates walnut genotypes presenting relatively high susceptibility in the field (percentage of necrotic leaf area > 1.25) to G. leptostyla.

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Table 3 Number of amplified fragments (total, private, common) and level of polymorphism for each enzyme–primer combination used for NBS profiling analysis of 41 J. nigra and 31 J. regia genotypes collected in the Azienda Ovile site. Enzyme–NBS primer combination

Size range (bp)

Number of amplified bands Total

MseI–NBS1 RsaI–NBS1 MseI–NBS5-A6 RsaI–NBS5-A6 Total

63–602 82–640 84–610 85–570

117 82 85 33 317

Number of polymorphic bands

Species-private J. nigra

J. regia

48 36 42 19 145

43 32 39 11 125

Common

Total

26 14 4 3 47

101 72 81 31 285

Species-private

Common

J. nigra

J. regia

27 27 28 11 93

21 14 26 6 67

10 4 – 1 15

Table 4 Diversity parameters for neutral SSR and functional NBS markers detected in 41 black walnut and 31 Persian walnut trees: effective number of SSR alleles or NBS bands (Ne ), Nei’s (1973) gene diversity index (He ), Shannon’s diversity index (I), and their standard deviation (number in parenthesis), percentage of polymorphism (P), Power of Discrimination (PD), FST genetic differentiation among populations according to Weir and Cockerham (1984), RST genetic differentiation among populations according to ), estimator of actual differentiation Dest , (Jost, 2008) and analogous FST coefficient (Excoffier et al., 1992) for binary data. Neutral SSR markers Ne Juglans nigra L. 4.070 (1.944) 2.712 (0.647) Juglans regia L. Average 5.659 (1.653) Functional NBS markers Ne Juglans nigra L. 1.165 (0.297) 1.134 (0.285) Juglans regia L. 1.585 (0.394) Average a b *

He

I

Pb

PD

FST a

RST

Dest

0.688 (0.668) 0.609 (0.107) 0.8092 (0.056)

1.535 (0.457) 1.083 (0.288) 1.929 (0.265)

100.0 100.0 100.0

0.974 0.967 0.990

0.315*

0.928*

0.871

He 0.101 (0.166) 0.078 (0.159) 0.322 (0.191)

I 0.153 (0.244) 0.118 (0.231) 0.471 (0.254)

P

PD 0.966 0.943 0.970

54.10 46.30 89.90b

FPT

0.864*

Level of significance of genetic differentiation indices were tested using a non-parametric approach described in Excoffier et al. (1992) with 1000 permutations. Polymorphism level was calculated including private-species and common NBS bands. P < 0.01.

with any reported plant resistance or resistance analogs genes. BLAST search from the public non-redundant (nr) Viridiplantae database (GenBank database http//www.ncbi.nlm.nih.gov) also found no similarity with plant ESTs.

4. Discussion 4.1. Disease avoidance by late flushing In this study we analyzed 72 walnut genotypes showing a differential response towards G. leptostyla infection in the field recorded during the summers of 2004 (Annunziati et al., 2007) and 2008 (Table 1). A statistically significant correlation was found between anthracnose resistance in the field and the leaf emergence rate of the trees. All late flushing J. nigra plants (JN001–JN126), except for the JN087 genotype, were significantly resistant to anthracnose when compared to the 31 early flushing J. regia genotypes (RA004–RA128). As suggested by Ghelardini and Santini (2009), early or late flushing represents a mechanism of disease avoidance based on the temporal separation of susceptible stages in the life cycle of the host and the favourable environmental conditions for the rapid proliferation and development of the pathogen. Belisario et al. (2001) reported that G. leptostyla usually overwinters as ascocarps in fallen leaves, rarely as mycelium on the lesions of twigs and fruit. In central Italy (Rome) primary infection occurs in the early spring through ascospores released from perithecia (Belisario et al., 2008). If they lodge on a susceptible leaf under optimal environment conditions (abundant rain and leaf wetness), the ascospores germinate and infect the leaf. Several secondary infections occur during the growing season by conidia produced in black, translucent, fruiting bodies called acervuli (Belisario et al., 2001). Our data revealed that during the second half of April 2008 (Jd 93), when the ascopores should be released, approximately 25% of the black walnut genotypes had completely closed buds and the

remaining 75% had young leaves not exceeding stage 3 (emerging leaves < 2 cm). We can assume that trees in which buds remain closed at the beginning of first sporulation are less prone to disease, since susceptible tissues are not present. In addition, it has been clearly demonstrated by Cline and Neely (1984) that young, succulent walnut leaves are less susceptible to infection than fully expanded leaves. Therefore during primary infection, high levels of juglone in juvenile leaves of J. nigra trees may induce delayed germination of ascospores and ultimately prevent pathogen development (germination and penetration). Conversely, as reported in Table 1, J. regia genotypes whose leaves had emerged early were most likely infected by G. leptostyla. On 14 April 2008, 67.74% and 22.5% of the J. regia trees showed expanding shoots <4 cm (stage 5) and shoots longer than 4 cm, respectively. As explained by Jacobs and Danielson (2002) for Fraxinus spp. “a longer exposure time of susceptible tissue to inoculum can expand epidemics of polycyclic diseases such as anthracnose by allowing secondary infection and conidia production to occur earlier and in greater abundance”. In Persian walnut trees, the rate of epidemic progress in the field may be more influenced by the dispersal ability of the pathogen than in J. nigra trees (Frantzen, 2000). Thus, we postulated that under the selection pressure imposed by G. leptostyla, late flushing J. nigra plants may have an advantage, by avoiding the impact of anthracnose disease. This escape mechanism is defined as avoidance by late flushing. However, in this case infection and subsequent disease development are prevented through the presence of temporal barriers. As explained by Frantzen (2000), after the direct inoculation of fungus in the target tissues, plants with a disease avoidance mechanism and no host specific resistance should be infected. In our study, pathogenicity tests by the direct application of the ISPaVe2000 strain of G. leptostyla in the fully expanded leaves of the two-year old clones of each walnut genotype enabled us to discriminate partially resistant from susceptible genotypes and

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genotypes selected in our experimental plantation were much more resistant than J. regia genotypes. The ISPaVe2000 strain of G. leptostyla was isolated from leaves of ‘Lara’ variety collected in a Persian walnut orchard (Monterotondo, Rome) during summer 2008 (CRA-PAV3033; http://www.colmia.it/bancaDati.cfm). Thus we may postulate some kind of co-evolution between Persian walnut plants and the ISPaVe2000 strain of G. leptostyla. Finally, our results indicate that a partial host resistance mechanism may also play a crucial role in restricting damage by G. leptostyla. These data led us to assume that resistance to this pathogen in the field should be considered as a quantitative trait and possibly poly-genetically inherited. 4.2. Partial host resistance mechanism

Fig. 6. A section of NBS profiling LI-COR image obtained using NBS1/RsaI as a primer/restriction enzyme combination. Nine J. regia genotypes (lines 1–9) and one J. nigra genotype (line 10) with relatively high in vivo susceptibility (percentage of necrotic leaf area > 1.24) and twelve J. nigra genotypes (lines 11–22) with in vivo partial resistance (percentage of necrotic leaf area < 0.52) to G. leptostyla are shown. Arrows indicate the position of polymorphism (fragment = 91 bp) potentially associated with an increase of susceptibility to walnut anthracnose.

revealed different putative levels of susceptibility to anthracnose. Forty J. nigra plants (JN001–JN126) classified as resistant in the field were infected in vivo. Inoculation in 92.5% of these black walnut genotypes caused symptoms that were more severe (about 2.5 times more susceptible) in comparison to those observed in the field. These discrepancies could be the result of disease avoidance by a late-flushing mechanism. However, we can not rule out that other factors, aside from the disease avoidance mechanism, could affect the expression of resistance components in the field, such as fluctuations in temperature, relative humidity, leaf moisture and inoculum concentration (these factors are currently under evaluation). On the other hand, 31 J. regia (RA004–RA128) and one J. nigra (JN087) plants that presented susceptibility in the field continued to be one order of magnitude more affected by anthracnose under artificial infection than the above mentioned J. nigra trees. Thus 31 J. regia (RA004–RA128) and one J. nigra (JN087) were classified as in vivosusceptible plants. By definition, a partial-resistant genotype leads to limited but significantly reduced pathogen reproduction when compared to a susceptible genotype (Ballini et al., 2008). Therefore the corresponding 40 J. nigra plants (JN001–JN126) were defined as in vivo partially resistant plants. Although black walnut trees were generally attacked by G. leptostyla in the U.S. (Woeste and Beineke, 2001) and only a few resistant J. nigra cultivars have been documented (e.g. Ohio, Thomas), nearly all J. nigra

The application of an NBS profiling technique that specifically targets conserved motifs in the functional NBS domain of receptorlike R-gene family members enabled us to display genetic variation in R genes and R-gene analogs (RGAs). Several studies in potato (Wang et al., 2008; Brugmans et al., 2008), lettuce, barley, tomato (Van der Linden et al., 2004), and apple (Calenge et al., 2005) demonstrated that the majority of fragments amplified were RGA derived, showing significant levels of similarity with known NBS domain sequences present in R-genes and RGAs of several species. In this study we used two degenerate NBS primers (NBS1, NBS5A6) and two restriction enzymes (MseI, RsaI) for a total of four NBS primer/enzyme combinations. This was done in order to amplify a multi-locus RGA marker pattern from genomic DNA of 40 J. nigra partial-resistant genotypes and 32 genotype susceptible (31 J. regia, 1 J. nigra) to anthracnose without any modification to the NBS primer sequences already applied in the above mentioned crop species. Our data confirmed the high level of transferability of NBS markers across species and indicated that they may represent an important tool for improvement strategies not only in Juglans but also in other forest trees. As reported by Van der Linden et al. (2004), NBS markers derived from transcribed regions of the DNA are generally more conserved across species. These functional markers are expected to have a higher rate of transferability than neutral markers such as SSRs. In contrast, the functional constraints on transcribed portions of the genome may result in a lower mutation rate than in the non-coding, non-transcribed regions, thus limiting the levels of polymorphism (Woodhead et al., 2005). Our results supported the above observations. In fact, measures of Nei’s (1973) gene diversity index (He ) and Shannon’s diversity index (I), were both higher for neutral SSR markers than for functional NBS markers. Although the NBS gene diversity in each species was moderately high, the probability that two unrelated trees drawn at random from J. nigra and J. regia groups would have identical genotypes at multiple loci is almost null for both NBS and SSR markers. In addition despite the non-neutral nature of NBS profiling markers, a high correlation was found between NBS and SSR individual pairwise genetic similarity values computed between the 72 walnut genotypes. The high level of genetic differentiation among Juglans species calculated in this study using the NBS approach (FPT = 0.864) and the congruency between SSR and NBS profiling is not unexpected and was consistent with data obtained in wheat (Mantovani et al., 2006) and Solanum species (Wang et al., 2008). Our results may partly depend on the high phylogenetic distance between black and Persian walnut. Phylogenetic analysis based on nuclear RFLP, matK and ITS sequence has demonstrated that J. nigra and J. regia belong to different sections of genus Juglans, Rhysocaryon and Dioscaryon respectively (Stanford et al., 2000). As suggested by Wang et al. (2008), species in the same genus showing a distinct indigenous area and geographic distribution have to deal with many different pathogens during their lifetime and are subjected to selective pressures in different directions.

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Nevertheless, Mantovani et al. (2006) questioned the use of genomic DNA as starting material for the NBS procedure, as it does not enable us to distinguish between expressed genes and silent genes or pseudo-genes characterized by a higher mutational rate. Other studies have demonstrated that a reliable number of NBSLRR genes were not expressed in sequence tag (EST) libraries of wheat (McFadden et al., 2006) and rice (Monosi et al., 2004). In potato, performing NBS profiling on cDNA extracted from different tissues led to the amplification of approximately 50% of the bands detected using genomic DNA as a template (Brugmans et al., 2008). In the future, the use of cDNA as a template for NBS profiling analysis may enable a selection against markers derived from non-expressed genes. The application of the NBS marker methodology on the 72 walnut genotypes revealed the presence of a candidate NBS marker potentially related to anthracnose responses in walnut. No amplification for two fragments of 91 bp in length using the NBS1 primer–RsaI restriction enzyme combination (NBS1–RsaI-91bpA and NBS1–RsaI-91bpB) appeared to be correlated to a reduction in anthracnose infection within 72 walnut genotypes. The DNA sequence of these fragments showed no significant homology with any known plant R gene or RGAs in crop species. The NBS markers of interest were amplified using genomic DNA and may not be derived from expressed genes. Nevertheless, taking into account the short length of the candidate markers and the relative lack of information regarding R and RGA gene sequences in forest tree species, we cannot completely rule out that our candidate marker does not belong to the family of NBS-LRR genes. In addition, it should be considered that the NBS1 primer amplified DNA towards the 5 end of targeted genes, thus in less conserved regions outside the NBS domain. The association between NBS-LRR genes and QTLs conferring resistance to anthracnose mainly caused by Colletotrichum spp. pathogens has already been reported in other plant species such as Medicago truncatula (Yang et al., 2007; Ameline-Torregrosa et al., 2008), Lupinus angustifolius (You et al., 2005) and Phaseolus vulgaris (Ferrier-Cana et al., 2003). In alfalfa the high-resolution genetic and physical mapping delimited the RTC1 locus for anthracnose resistance by Colletotrichum trifolii within a physical spanning of ∼200 kb located on the top of linkage group 4. Yang et al. (2007) revealed that RTC1 was part of a complex locus containing three complete TIR-NBSLRR genes. In an earlier study, Ameline-Torregrosa et al. (2008) also demonstrated the presence of 25 RGA, mainly showing an atypical structural organization in the upper part of chromosome 4. In L. angustifolius, the application of the MFLP approach and the replacement of SSR-anchor primers with NBS primers designed on three different conserved motifs (P-loop, kinase2, GLPL) enabled a 217 bp marker (AntjM2) tagging the anthracnose resistance gene Lnr1 to be developed (You et al., 2005). As with our findings, the DNA sequence of the latter fragment showed no significant homology with any nuclear DNA, EST or encoding sequence in GenBank. In the literature the major detected QTLs were always associated with dominant R-gene-based complete resistance of a host to an anthracnose pathogen. This type of resistance is generally assumed to be race specific and not durable because of the extensive diversity displayed by the pathogen. On the other hand, it was frequently assumed that partial resistance, resulting in a limited epidemic build-up, is conditioned by genes with small effects and should not be based on receptor–elicitor recognition (McDonald and Linde, 2002). Over the last decade several studies have challenged the widely held models by finding that partial resistance can involve not only general defence mechanisms but also can be R-gene-related (Poland et al., 2008). The QTL co-localization of partial resistance against Colletotrichum lindemuthianum in common bean with cloned defence genes (ex. hydroxyproline-rich glycoprotein),

resistance analogs genes (RGAs) and anthracnose-specific resistance genes involved in pathogen recognition (NBS-LRR) has been already demonstrated (Geffroy et al., 2000). Durel et al. (2003) proposed that specific partial resistance could be controlled by defeated R-genes. According to the residual resistance-model, once overcome by a pathogen strain, R-genes may conserve some residual effect. Similarly, the walnut phenotypic variance observed in our study can be explained by the weaker form of the R-genemediated defence mechanism. The partially resistant phenotypes of black walnut showing restricted leaf lesions in the pathogenicity tests, might be a result of the inadequate recognition of the G. leptostyla elicitor by the weak-effect R-gene. Nevertheless, as suggested by Poland et al. (2008), it is also possible that other mechanisms might be involved in quantitative partial resistance of plant, such as a minor-gene-for-minor-gene interaction where a specific resistance gene of minor effect in the host recognizes the product of a virulence gene of minor effect in the pathogen. Recently two recessive genes (pi21, pi34), without any nucleotide similarity to any current known defence-related genes, have been found to regulate partial resistance towards blast disease in rice (Ballini et al., 2008). 5. Conclusions Over the last decade there has been an increasing interest in the identification, selection and production of genotypes resistant to anthracnose caused by G. leptostyla in Juglans spp. Unfortunately, several aspects of Juglans flower biology, such as low pollen viability and vitality and pistillate flower abscission (PFA) caused by excessive pollen load, have frustrated the production of controlled crosses and limited the progress of research and plant breeding (Pollegioni et al., 2010). However, the extensive phenotypic data of the last twenty years collected in black and Persian walnut plantations may have great potential for studying the mechanisms of anthracnose resistance in the field. In this study, the phenotypic data detected in the field offered the opportunity to dissect some of the mechanisms governing the responses to walnut anthracnose. Our results indicated that the incidence of anthracnose in the field is conditioned by two key factors namely the age and/or the availability of susceptible leaves during the first fungus sporulation time and the partial host resistance. On the basis of this preliminary evidence, the late-flushing walnut clones already selected in breeding programs to avoid other problems such as spring frost damage, may have a natural ability to avoid G. leptostyla infection. The development of sequence-specific PCR markers from the above mentioned NBS candidate fragment, the use of cDNA as starting material for the NBS procedure, and a larger number of walnut genotypes in NBS-profiling analysis may provide additional and valuable insights regarding the molecular mechanisms underlying the partial resistance against G. leptostyla in Juglans spp. Role of the funding source Paola Pollegioni was supported by a PhD fellowship in Forest Ecology from the University of Tuscia (Viterbo, Italy). Part of this work was developed in the framework of the Italian MiPAAF Project JUGL’ONE (Ricerca e sperimentazione florovivaistica multidisciplinare per la costruzione di ibridi di noce Italiani polifunzionali). Acknowledgements The authors would like to thank Hanneke van der Schoot, Marcello Cherubini and Daniela Taurchini for their contribution to the lab work, Eric van de Weg and Anitha Kumari for stimulating discussions, Giovanni Mughini, Manuela Annunziati for providing

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