apple proliferation disease in croatian orchards - SIPaV

Journal of Plant Pathology (2017), 99 (1), 95-101 Edizioni ETS Pisa, 2017 95

APPLE PROLIFERATION DISEASE IN CROATIAN ORCHARDS: A MOLECULAR CHARACTERIZATION OF ‘CANDIDATUS PHYTOPLASMA MALI’ I. Križanac1, J. Plavec1, Ž. Budinšcák1, D. Ivic´1, D. Škoric´2 and M. Šeruga Music´2 1 Institute

for Plant Protection, Croatian Centre for Agriculture, Food and Rural Affairs, Gorice 68 b, HR-10000 Zagreb, Croatia of Biology, Faculty of Science, University of Zagreb, Marulicév trg 9A, HR-10000 Zagreb, Croatia

2 Department

SUMMARY

INTRODUCTION

Apple proliferation (AP) causes major economic losses to the European apple industry. The causal agent associated with AP is ‘Candidatus Phytoplasma mali’ which belongs to the same phylogenetic cluster as two other fruit tree phytoplasmas: ‘Ca. P. pyri’ and ‘Ca. P. prunorum’. ‘Ca. P. pyri’ and ‘Ca. P. solani’ have sporadically been associated with symptomatic apples in Croatia in previous surveys. There was no molecular evidence of ‘Ca. P. mali’ presence until the start of this study, when it was confirmed for the first time. In this four-year survey (2011-2014), the detection and characterization of ‘Ca. P. mali’ from apples and psyllid vectors was carried out. Detection was based on PCR-RFLP analysis of the 16S rRNA gene. Real-time PCR was also used for amplifying ‘Ca. P. mali’ 16S rDNA and Malus domestica chloroplast tRNA leucine gene. Phylogenetic analysis of 16S rDNA and aceF gene sequences confirmed the identification of selected phytoplasma strains. Considerable aceF variability among the strains was detected, and a new, yet unrecorded aceF genotype was found. ‘Ca. P. mali’ has consistently been identified with high incidence in apple samples from Croatian continental region orchards, whereas it occurred sporadically in the coastal areas. This is the first molecular detection and characterization of ‘Ca. P. mali’ strains from both apple (Malus domestica Burkh.) and psyllid vector Cacopsylla picta (Förster) in Croatia.

Phytoplasmas induce diseases in more than a thousand plant species worldwide, many of which are of great economic importance. Their complex life cycle involves colonization of both plant hosts and insect vectors, as both are necessary for dispersal and long-term survival in nature (Marcone, 2014). These wall-less plant pathogenic prokaryotes belong to a monophyletic clade of the class Mollicutes. Their phylogeny is mainly based on conserved genes sequence analysis, the common bacterial phylogenetic marker 16S rRNA gene in particular (IRPCM, 2004). In addition, several non-ribosomal genes and multilocus sequence typing (MLST) are used to facilitate the differentiation of phytoplasma strains, trace their propagation routes, elucidate their molecular epidemiology and improve disease control (Urwin and Maiden, 2003; Danet et al., 2011). Apple proliferation (AP) causes major economic losses to the European apple industry. Typical symptoms are witches’ brooms, enlarged stipules and reduced fruit size and quality (Seemüller, 1990). The AP causal agent, ‘Candidatus Phytoplasma mali’, along with two other fruit tree phytoplasmas occurring in Europe, ‘Ca. P. pyri’ and ‘Ca. P. prunorum’, belongs to the same phylogenetic cluster (Seemüller and Schneider, 2004). Their chromosomes are linear, which is an unusual feature for phytoplasmas and bacteria, in general (Kube et al., 2008). Two psyllid species are identified as vectors of ‘Ca. P. mali’: Cacopsylla picta (Förster) [syn. C. costalis (Flor)] (Frisinghelli et al., 2000; Jarausch et al., 2003) and C. melanoneura (Förster) (Tedeschi et al., 2002). One leafhopper species, Fieberiella flori (Stål), was reported as a vector in north-western Italy (Tedeschi and Alma, 2006). The frequency of naturally infected C. picta and C. melanoneura, as well as their ability to transmit the disease, differs significantly in countries and regions where the disease is present and well studied (Jarausch et al., 2011; Mayer et al., 2009; Tedeschi et al., 2003). In 2008, a three-year inventory of grapevine and fruit tree phytoplasma vectors in Croatia was completed. Both C. melanoneura and C. picta were found to be present and widespread. Although C. melanoneura populations are more numerous, C. picta adults are present in apple orchards for a longer period, which increases their potential for phytoplasma acquisition (Budinšcák, 2008).

Keywords: 16S rDNA, aceF, variability, phylogeny, psyllids

Corresponding author: M. Šeruga Music´ E-mail: [email protected]

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Journal of Plant Pathology (2017), 99 (1), 95-101

Apple proliferation in Croatia

Table 1. Number of samples tested per year by regions and hosts. Region

Host

No. positives (No. tested) 2011

2012

2013

Apple 5 (16) Continental east Cacopsylla melanoneura − (CE) Cacopsylla picta −

1 (16) 0 (1) 0 (2)

5 (18) 0 (11) − − − −

1 (19)

5 (18) 0 (11)

Subtotal

5 (16)

2014

Apple 7 (28) Continental west Cacopsylla melanoneura − (CW) Cacopsylla picta −

8 (26) 24 (37) 8 (13) − 0 (4) − − 1 (2) −

Subtotal

7 (28)

8 (26) 25 (43) 8 (13)

2 (8)

0 (10)

0 (6)

0 (3)

2 (8)

0 (10)

0 (6)

0 (3)

Adriatic (AD)

Apple

Subtotal Subtotal – all regions Total – all regions in 4 years

14 (52)

9 (55) 30 (67) 8 (27) 61 (201)

The occurrence of AP in Croatian apple trees, based on symptomatology and the presence of phytoplasma cells in leaf mid vein sieve tubes, was first reported by Šaric´ and Cvjetkovic´ (1985). The presence and diversity of fruit tree phytoplasmas and their psyllid vectors in Croatia has also been investigated in several surveys over last 10 years. ‘Ca. P. pyri’ and ‘Ca. P. prunorum’ were detected in their respective host plants and vectors (Križanac et al., 2010) and the genetic diversity of several Croatian isolates was analysed (Danet et al., 2011). The presence of ‘Ca. P. pyri’ and ‘Ca. P. solani’ was sporadically associated with symptomatic apples, but there was no molecular evidence of the presence of ‘Ca. P. mali’ in diseased commercially grown apple trees and psyllid vectors (Križanac et al., 2010). The preliminary results on the molecular identification of ‘Ca. P. mali’ in Croatia were reported on a limited number of samples from 2011 (Plavec et al., 2013). In this study, the molecular identification and characterization of ‘Ca. P. mali’ in apple trees and of Cacopsylla sp. collected in Croatian orchards is reported for the complete survey period (2011-2014), along with data on phytoplasma geographical distribution. MATERIALS AND METHODS

Sampling. In a four-year period (2011-2014), commercial apple orchards in continental west (CW, 12 locations), continental east (CE, 5 locations) and Adriatic (AD, 4 locations) fruit-growing regions of Croatia were surveyed for AP symptoms. Apart from agro-climate differences, these regions differ somewhat in orchard management and presence of other apple common pests and diseases. Apple leaves and petioles were collected from September to mid-October, i.e. the optimal period for AP sampling (OEPP/EPPO, 2016), preferentially from trees displaying typical AP-like symptoms (witches’ broom, enlarged peduncles and small-sized fruits). However, to

perform a thorough survey and detect latent AP infections or apple trees infected early in the season at locations where AP-like symptoms were not observed, trees showing atypical symptoms (leaf reddening and apical rosette formation) or non-symptomatic trees were also randomly sampled. Psyllids were collected in 2012 and 2013 using the beating method (Steiner, 1967) at locations where ‘Ca. P. mali’ had been identified in apples in previous years (Osijek and Sveta Marija). Psyllids sampling was done in commercial orchards during March and April, depending on the year. Only overwintering psyllids were collected before the application of insecticides. Altogether, 192 samples of different apple cultivars and nine individuals of psyllid vectors were collected and tested (Table 1). DNA extraction. Leaf midribs and individual psyllids were used for total nucleic acids extraction following the procedure described by Angelini et al. (2001) with modifications introduced by Šeruga et al. (2003). DNA concentration and purity was measured using a Genova Nano microvolume spectrophotometer (Jenway, UK). Dilutions of 10, 20 and 100 ng DNA/µl were used in PCR/RFLP and real-time PCR experiments. Phytoplasma detection and identification. PCR/RFLP analyses of phytoplasma 16S rDNA were performed using P1/P7 primers in direct PCR assays (Deng and Hiruki, 1991; Schneider et al., 1995) followed by a nested PCR using R16(X) F1/R1 primers (Lee et al., 1994). PCR products were separated in 1% agarose gel, stained with Olerup GelRed solution (Olerup SSP, Sweden) and visualized under UV light using a gel documentation system (UVIdoc, Uvitec, UK). Amplicons from nested PCR were digested with RsaI and SspI (Takara Bio, Japan) following the manufacturer’s instructions, to determine the affiliation to ‘Ca. P. mali’. Digested amplicons were separated in 2.5% agarose gel and visualized using the procedure described above. Apple proliferation reference strain DNA (AP15) used for comparisons was obtained from the phytoplasma collection of the DiSTA Phytoplasmology laboratory, University of Bologna, Italy (Bertaccini, 2010). In order to increase the sensitivity of detection and confirm the results of conventional PCR/RFLP, real-time PCR experiments amplifying ‘Ca. P. mali’ 16S rDNA and the Malus domestica chloroplast tRNA leucine gene were performed for all apple samples (Baric and Dalla-Via, 2004). Molecular characterization and phylogenetic analyses. The two 16S rDNA PCR products around 1.8 kb in size primed by P1/P7 primers from apple and insect samples were custom sequenced on both strands (Macrogen, The Netherlands) to confirm the ‘Ca. P. mali’ identity. Raw nucleotide sequences were assembled and edited using the Sequencher™ 4.7 software (http://www.genecodes.com/), then aligned with ClustalX 2.0 (Thompson et al., 1997).


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Table 2. List of samples tested positive for the presence of ‘Ca. P. mali’ by region and sampling location together with GenBank accession numbers for the aceF gene sequence (where applicable). The region codes are the same as in Table 1. (*) marks the ‘Ca. P. mali strains’ chosen for 16S rDNA sequencing. Region

Sample ID/YY

CW

Host: Apple 472/13* 473/13 474/13 475/13 477/13 276/14 268/14 436/13 437/13 438/13 250/14 420/11 441/13 252/14 489/11 490/11 364/12 373/13 460/13 277/14 278/14 279/14 280/14 461/13

AD

CE

GenBank Acc.No.

KU644717

KU644729 KU644722

Sampling location

Sample ID/YY

GenBank Acc.No.

Sampling location

Donji Mihaljevec Donji Mihaljevec Donji Mihaljevec Donji Mihaljevec Donji Mihaljevec Donji Mihaljevec Katoličko Selišće Mičevec Mičevec Mičevec Mičevec Šušnjari Šušnjari Šušnjari Velika Ludina Velika Ludina Velika Ludina Velika Ludina Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija

481/11 483/11 519/11 522/11 310/12 311/12 389/12 390/12 391/12 392/12 393/12 448/13 449/13 450/13 451/13 452/13 453/13 454/13 455/13 456/13 457/13 458/13 459/13

KU644716 KU644728

Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija Sv. Marija

Host: Cacopsylla picta 6K/13* KT325594

Sv. Marija

Host: Apple 493/11 500/11

KU644715

Crepina Komin

KU644726 KU644725 KU644724 KU644723

Osijek Osijek Osijek Osijek Osijek Osijek

Host: Apple 354/11 355/11 356/11 357/11 391OS/13 411/13

Phylogenetic analyses were performed with the MEGA 5 software (Tamura et al., 2011) using the neighbour-joining method with number of differences model. Bootstrap analysis (500 replicates) was done to estimate the stability of nodes and to support the inferred clades (Felsenstein, 1985). Furthermore, the entire aceF gene encoding pyruvate dehydrogenase (dihydrolipoamide acetyltransferase component) was amplified in a direct PCR using newly designed primers (acoB_F: 5’-CTGCTCCATCTAGAGTTAC-3’ and lpd_R0m: 5’-GCTAGCTTTTATAGCAGCT-3’) for assessing the genetic variability of ‘Ca. P. mali’ isolates. Amplicons of approximately 1.6 kb were amplified under the following PCR conditions: initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C/1 min, 52°C/ 2 min, 68°C/3 min and a final extension step of 7 min at 68°C. Sequencing, assembly and phylogenetic analyses of aceF gene sequences were performed as described for the 16S

368/12 397/13 399/13 554/11 425/13

KU644727

KU644720 KU644719 KU644718

KU644721

KU644714

Staro Petrovo selo Staro Petrovo selo Staro Petrovo selo Žubrica Žubrica

rDNA. All representative 16S rDNA and aceF sequences were deposited in GenBank. RESULTS

Prominent and typical AP symptoms such as witches’ brooms, enlarged stipules and serrated leaf blades, were observed regularly in all locations where ‘Ca. P. mali’ was identified, most notably in the continental west region. In two surveyed orchards from this region (Donji Mihaljevec and Sveta Marija), about 20% and 50% of the trees, respectively, were showing typical AP symptoms (Table 2). In the Adriatic region, atypical symptoms such as apical rosettes on less vigorous trees were observed. As previously reported (Seemüller et al., 2011), symptoms were more conspicuous in trees grafted on MM 106 than on M9 rootstock. However, witches’ brooms were also noted in

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including previously reported representative aceF sequences of ‘Ca. P. mali’ (Danet et al., 2011), revealed the presence of four different aceF genotypes among the Croatian samples, including a new, yet unreported genotype (Fig. 3) DISCUSSION

Fig. 1. Representative RFLP patterns of phytoplasma 16S rDNA amplified using R16(X) F1/R1 primers, digested with RsaI (lanes 1, 3, 5, 7, 9, 11, 13) and SspI (lanes 2, 4, 6, 8, 10, 12, 14) and separated in 2.5% agarose gel. Lanes 1-12, apple samples 2013: 373, 391OS, 397, 399, 437 and 438. Lanes 13-14: AP-15 reference strain. M9-PhiX174, HindIII digested (fragment sizes: 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118 bp).

trees growing on the less vigorous M9 rootstock. Smaller fruits with longer peduncles were clearly visible on most trees with AP symptoms, independently of the cultivar or rootstock. Seasonal variability in symptoms expression was evident at locations surveyed for several consecutive years. In 2011, the presence of ‘Ca. P. mali’ was confirmed in 14 of 52 samples collected from apple orchards at seven locations: Osijek and Žubrica in the CE region, Šušnjari, Sveta Marija and Velika Ludina in the CW and Crepina and Komin in the Adriatic region (Table 1 and 2). In subsequent years, AP continued to be observed in the surveyed locations of the continental part of the country, however, in the Adriatic region the disease was only found in the first year of the survey (Fig. 1; Table 1 and 2). Moreover, in 2013 and 2014 ‘Ca. P. mali’ was consistently detected in the majority of the apple samples from the CW region (Table 1 and 2). A very severe outbreak was observed in Sveta Marija, where in those two seasons 19 of 20, and 5 of 6 tested apple samples, respectively, proved to be AP positive along with the infected C. picta sample. The results obtained using both detection methods (PCR/RFLP and real-time PCR) were congruent, for all apple samples, except for 391/13, 397/13 and 437/13 (Table 2) where only real-time PCR was positive with threshold cycle values of 24, 33.1 and 26.2, respectively. Phylogenetic analysis of 16S rDNA representative sequences from both insect vector 6K (accession No. KJ676479) and apple sample 472 (KJ676480) (Table 2; Fig. 2) confirmed the affiliation to ‘Ca. P. mali’ with 99% ID to the sequence of ‘Ca. P. mali’ strain AP15 (AJ542541). Sequencing and phylogenetic analyses of the entire aceF gene showed a very close relatedness to the aceF gene sequence from the fully sequenced genome of the AT strain of ‘Ca. P. mali’ (CU469464) with 99% sequence identity for most of the obtained sequences (not shown). However, phylogenetic analyses of partial aceF gene sequence,

Apple proliferation is considered to be the most important graft-transmissible disease of apple in Europe. However, due to insufficient data, the overall economic impact of the disease in Croatia is difficult to estimate. In a four-year period (2011-2014), the highest incidence of trees with typical AP symptoms were observed in 2013 at Sveta Marija in the CW part of the country (Table 1 and 2). In this location, the incidence of symptomatic trees was permanently high during all four years of the survey. At other locations of the same region, the number of symptomatic trees was much lower, usually less than 5%, depending on the year, cultivar, rootstock or orchard. The CW part of the country, besides being an important apple-producing area, is apparently also the one with the most favourable conditions for both the disease and vector survival, resulting in highest AP pressure. In the Adriatic part of the country, AP symptoms were difficult to discern and symptomatic trees were observed sporadically only in two orchards in 2011. In the CW Croatia, the detection of nearly 50% of APpositive samples over the years indicates that the disease pressure in this important apple production area is high. Apple samples in which the presence of phytoplasma cells had been demonstrated in 1985 by Šaric´ and Cvjetković (1985) were also from this region, indicating that AP has been present in the area for several decades. However, AP incidence may be underestimated in the other two regions (CE and AD) considering a possible influence of seasonal fluctuations in symptom manifestation or remittance (Seemüller et al., 1984; Carraro et al., 2004), as well as a variable symptom expression in different cultivars (Lešnik et al., 2007) and the potential presence of latent infections (Baric et al., 2007). The first finding of psyllids actually carrying the phytoplasma in the CW part of the country is a confirmation that infective AP vectors are present in the field. A low number of collected psyllids in the surveyed areas may be a consequence of a short time frame suitable for sampling of overwintering psyllids before the first application of insecticides in commercial orchards. Different phytoplasmas infecting wild apples and pears were recently reported in continental Croatia (Ježic´ et al., 2016), demonstrating the presence of AP reservoirs outside the commercial orchards. Altogether, this suggests that more studies on AP incidence, severity, vector presence and population dynamics should be carried out before control measures of Cacopsylla spp. are recommended.


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Fig. 2. Phylogenetic tree inferred using neighbour-joining (NJ) analysis of phytoplasma 16S rRNA gene sequences. The phytoplasma strains used in the phylogenetic tree construction include representative members of different 16S groups with Acholeplasma palmae as an outgroup. Numbers above branches indicate bootstrap values above 50 (500 replicates). Bar length represents nucleotide changes. Sequences of 6K (GenBank accession No. KJ676479) and 472 (KJ676480) group together in a separate branch with ‘Candidatus Phytoplasma mali’ reference strain (CU469464).

The results of AP detection and identification based on 16S rDNA obtained using both PCR/RFLP and the very sensitive real-time PCR were congruent (Fig. 1; Table 1 and 2) with the exception of three apple samples where only real-time PCR was positive. Correspondingly, for two of these samples (397/13 and 437/13), amplicons were not obtained in direct PCR using newly designed primers amplifying aceF gene. This may indicate that either the pathogen was present in a low titre in sample 397/13, as suggested by the higher threshold cycle value in the real-time PCR detection, or that nucleotide changes had occurred in the aceF gene region (sample 437/13). This discrepancy in the results of testing needs to be elucidated further. Newly designed primers from this study enabled the amplification of the entire aceF gene. Previously, a fragment of this gene, together with imp, secY and pnp gene fragments was used in a multilocus sequence analysis of European fruit tree phytoplasmas (Danet et al., 2011) where six different aceF genotypes of ‘Ca. P. mali’ were reported. The phylogenetic analyses of aceF gene from this study revealed a considerable variability and the presence of four different genotypes among the analyzed Croatian

AP strains (Fig. 3). According to Danet et al. (2011), the majority of samples (12 out of 17) corresponded to the A13 aceF genotype that was previously detected in Germany, Italy and France. Two samples corresponded to the A16 genotype, while only one was identical to the A15 genotype reported from France, Italy and Austria. A new SNP was found in aceF gene fragment from 310/12 and 481/11 samples revealing the presence of a new yet unreported genotype (Fig. 3). Additionally, our results further corroborated the significance of aceF gene as a valuable molecular marker in multilocus gene typing as a tool for molecular epidemiology studies. Further monitoring of vectors, in particular in abandoned orchards or wild hosts, is needed for setting up effective and environmentally friendly psyllid control strategy. During the survey, it was noticed that Croatian apple growers often do not recognize AP symptoms and are not familiar with the potential impact of the disease. Increasing the awareness of AP disease impact among Croatian apple producers could also contribute to the further improvement of apple production in Croatia.

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Fig. 3. Unrooted phylogenetic tree inferred using neighbourjoining (NJ) analysis of partial phytoplasma aceF gene sequences of ‘Candidatus Phytoplasma mali’ and attributed aceF genotypes according to Danet et al. (2011). Numbers above branches indicate bootstrap values (500 replicates). Bar length represents nucleotide changes. GenBank accession numbers of reference sequences are given next to the taxon name while the accession numbers of samples are given in Table 2 next to the sample ID. ACKNOWLEDGEMENTS

This research was funded by the Ministry of Agriculture, Croatian Centre for Agriculture, Food and Rural Affairs and partially by the University of Zagreb (grant 202664) and the Croatian Science Foundation (grant UIP2014-09-9744). Authors wish to thank Jelena Norac-Kljajo for her technical assistance. REFERENCES Angelini E., Clair D., Borgo M., Bertaccini A., Boudon-Padieu E., 2001. Flavescence dorée in France and Italy – Occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma. Vitis 40: 79-86. Baric S., Dalla-Via J., 2004. A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. Journal of Microbiological Methods 57: 135-145. Baric S., Kerschbamer C., Dalla-Via J., 2007. Detection of latent apple proliferation infection in two differently aged apple orchards in South Tyrol (northern Italy). Bulletin of Insectol-

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Received April 26, 2016 Accepted October 24, 2016

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