Protection Service from Badajoz), M. Cambra. (Plant Protection Service from Zaragoza), M. P.. Baraja (Plant Certification Service from Valencia) and T. Orero ...
Plant Pathology (1997) 46, 694–698
Pear decline in Spain ´ CER a , J. ALMACELLAS b and R. TORA ´b L. AVINENT a , G. LLA Instituto Valenciano de Investigaciones Agrarias, Apdo Oficial, 46113 Moncada, Valencia; and bServei de Proteccio´ dels Vegetals, Alcalde Rovira Roure 177, 25006 Lleida; Spain
a
Pear decline in Spain, associated with the presence of phytoplasmas in sieve tubes, was studied. Samples of healthy and diseased pear trees were tested to confirm the presence of the pathogen. The polymerase chain reaction (PCR) technique was used, with universal and specific primers. Specimens of Cacopsylla pyri were also analysed by PCR. Phytoplasmas were detected in 79% of trees with premature reddening, in 67% of trees with weakness and necrotic spots (cv. Limonera) and in 20% of trees without symptoms. The pathogen was also detected in the psyllids, indicating that C. pyri could be the vector of the disease in Spain.
INTRODUCTION Pear decline is a disease associated with the presence of phytoplasmas (formerly MLOs) in the sieve tubes (Hibino & Schneider, 1970). It is known from the United States, Canada (McLarty, 1948), and several European countries: Austria, Belgium, the former Czechoslovakia, England, France, Germany, Greece, Italy, Rumania, Switzerland and the former URSS (Kristensen, 1976; Nemeth, 1986; Davies et al., 1992). The psyllid Cacopsylla pyricola is responsible for spreading the disease in North America (Jensen et al., 1964; Hibino et al., 1971) and England (Davies et al., 1992). In Spain, pear decline is thought to have been present since the 1960s, according to some studies on tree symptomatology but the pathogen was not studied (Rallo, 1973; Pen˜a-Iglesias, 1977). In recent years many declining pear trees have been detected in Spain. Trees of most varieties showed early leaf reddening, sometimes as early in the year as July, red bark, weak foliation, slight leaf rolling and death of the tree. Trees of cv. Limonera showed some variations: leaf chlorosis, necrotic spots on leaves and strong defoliation in summer. Tests for fungi, bacteria, virus or cultural disorders as the cause of the decline were always negative. In 1993 studies to detect phytoplasmas using the polymerase chain reaction (PCR) technique were initiated in Spain. Phytoplasmas were detected in pear trees showing symptoms of pear decline while no pathogen was detected in healthy trees (Avinent & Lla´cer, 1994). These preliminary studies led to the conclusion that pear decline, as a disease associated Accepted 11 April 1997.
with phytoplasmas, was present in Spain but no data on disease incidence were available. The present study seeks to widen knowledge of the disease in Spain. MATERIALS AND METHODS Pear tree samples Samples were collected mainly in autumn 1994 and 1995 from the following locations: (a) Lleida and Zaragoza: the two main areas for pear growing in Spain, samples were from either commercial fields or nurseries; (b) Badajoz: the fourth most important location for pear growing, samples were from commercial fields; (c) Sevilla and Valencia: minor locations for pear growing but having important nurseries from which the samples were collected. The number of pear trees analysed, according to symptoms observed is shown in Table 1.
Insect samples Samples of Cacopsylla pyri, the pear psyllid, were collected from plots with or without symptoms in Lleida and Zaragoza, and sent for analysis. They were caught by the frappage method (hitting the tree, 40 strokes per tree, and collecting the falling insects on a spread cloth). Pear psyllids were separated from other fauna, placed in plastic bags with some pear tree leaves, and sent to the laboratory in Valencia. Insects that arrived alive were transferred to healthy Catharanthus roseus plants to check for transmission. Groups of 5–10 psyllids were confined to these plants by
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Table 1 Number of pear trees analysed by PCR
Symptoms Early leaf reddening, sometimes even in July, red bark, weak foliation, leaves slightly rolled
Leaf chlorosis, necrotic spots on leaves, strong defoliation in summer, general weakness and death of the tree. In some of these trees, some red foliage was obsvered at the end of 1995. Weak foliation in spring, trees dying in summer No symptoms of decline
Cultivars*
Number of Trees
Bartlett, Blanquilla, Conference, Ercolini, Abate Fetel, Williams, Sta. M. Morettini Limonera
43
Sta. M. Morettini Conference, Blanquilla, Ercolini, Sta. M. Morettini, Bartlett
Total number of pear trees analysed
24
8 20
95
*Rootstocks were: Pyrus communis, Cydonia oblonga and ownrooted plants.
introducing them into small cages (Fig. 1) enclosing one shoot or leaf of the plant per cage. Psyllids were kept there until they died, and were then tested by PCR for the presence of phytoplasma. Host plants were kept in the greenhouse and checked periodically for symptom expression. A total of 21 Catharanthus plants were used. The number of insects per plant ranged from 5 to 63, average 21. Insects lived from 1 to 25 days, and during this period they seemed to feed quite well on the plants. All these transmission trials were done in November 1995. Detection and characterization of the pathogen DNA was extracted from phloem tissue collected
from 1-year-old branch samples. Phloem was removed with a knife from the bark. DNA extraction and PCR amplification were done according to Ahrens & Seemu¨ller (1992). For the DNA amplification phytoplasma universal primer pairs (U5/U3, amplifying a fragment of about 880 bp in length) (Seemu¨ller et al., 1994) and more specific pome-fruit phytoplasma primer pairs (fPD/rO1, amplifying a fragment of about 930 bp in length) (Lorenz et al., 1995) were used. PCR reaction and visualization of results were done as previously described (Avinent & Lla´cer, 1994). In insects, DNA extraction was done as follows: groups of 5–10 insects were ground, using glass homogenizers, with 0.75 mL of CTAB extraction buffer (2% CTAB, 1.4 M NaCl, 0.2% 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0). Solution was transferred to 1.5-mL microfuge tubes and incubated at 608C for 30 min. The lysate was extracted with an equal volume of chloroform/ isoamyl alcohol (24:1 v/v) and centrifuged at 4000 r.p.m. for 5 min. Supernatant was precipitated with 0.5 mL of ¹208C isopropanol and centrifuged at 11 000 r.p.m. for 20 min. The pellet was washed with 70% ethanol, dried under vacuum and dissolved in 50 mL of distilled water. Primers used for amplification were fPD/rO1. Thirty-three groups of insects were analysed. Pathogen characterization
Fig. 1 Small cage for transmission on C. roseus.
For phytoplasma characterization, amplification of
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the 16S rRNA gene with primers U5/P7 (Seemu¨ller et al. 1994; Kirkpatrick et al., 1994) was performed. Amplified fragments, about 1400 bp in length, were subjected to restriction fragment length polymorphism (RFLPs) with AluI and RsaI restriction enzymes. PCR reaction and DNA digestion were done as described previously (Avinent & Lla´cer, 1994). RESULTS Pear trees were classified according to PCR results and symptoms observed (Table 2). Phytoplasmas were detected by PCR (Fig. 2) in trees showing symptoms of premature autumn reddening and/or weakness. The results (Table 2) showed phytoplasmas to be detectable in 34 out of 43 trees (79%) with reddening, in 16 out of 24 trees (67%) with weakness and necrotic spots (cv. Limonera) and in only four out of 20 symptomless trees (20%). Analyses of a 3 × 2 contingency table (data from Sta. M. Morettini were not included because the number of samples was very low) revealed that the association between symptoms and the presence of phytoplasmas was highly significant (x2 ¼ 20.5; P < 0.001). Positive trees were detected in samples from the five locations studied. Twenty-two pear samples were analysed with both pairs of primers fU5/rU3 and fPD/rO1 (Fig. 2). With fPD/rO1 four samples that had been negative with fU5/rU3 were positive, therefore increasing the number of positive reactions by 18%. Phytoplasmas were also detected by PCR (Fig. 3) in eight out of 33 psyllid samples (24%), from plots in either Zaragoza or Lleida. To the time of writing, none of the 21 host plants used in insect transmission had shown any symptom that could indicate a phytoplasma infection. Tests by PCR of these hosts were negative.
Fig. 2 Polymerase chain reaction amplification (PCR) of a 16S rDNA fragment from pear trees. Lanes 1–3, amplification with U5/U3: lane 1, Le´rida; lane 2, Zaragoza; lane 3, Badajoz. Lanes 4–8 amplification with PD/rO1: lane 4, Le´rida; lane 5, Zaragoza; lane 6, Valencia; lane 7, Sevilla; lane 8, Badajoz. Lane 9, molecular weight markers.
RFLPs of the fragments amplified with either U5/ P7 (1400 bp) or fPD/rO1 (930 bp) with AluI and RsaI from pear trees and psyllids revealed the same pattern for both types of samples (Figs 4, 5, 6 and 7). DISCUSSION Results indicate the role of phytoplasmas in the aetiology of pear decline and that the disease is present in all the pear tree-growing areas in Spain. In some infected plots in Zaragoza (Conference/ BA29) farmers have estimated that 15% of trees are infected when they are only 1-year-old increasing to
Table 2 Results of DNA amplification from pear-tree samples (either with fU5/rU3) and/or fPD/rO1) Number of trees Symptoms Reddening Weakness and necrotic spots (cv. Limonera) Weakness (cv. Sta. M. Morettini) No symptoms
PCRþ
PCR¹
34 16
9 8
3
5
4
16
Fig. 3 Polymerase chain reaction amplification (PCR) of a 16S rDNA fragment with PD/rO1. Lane 1, molecular weight markers; lanes 2 and 3, samples of C. pyri; lane 3, sample of a pear tree.
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Fig. 4 AluI restriction profiles of a 16S rDNA fragment amplified with U5/P7 from phytoplasmas found in different hosts. Lane 1, Diaphorina putonii; lane 2, plum tree; lanes 3 and 4, pear trees; lane 5, C. pyri; lane 6, no sample; lane 7, strawberry; lane 8, pepper; lane 9, molecular weight markers.
65% in the second year in the field. In Lleida, the Plant Protection Service has estimated that probably 80% of the plots checked are infected with pear decline, while number of infected trees per plot was variable. These data are very preliminary and an extensive and systematic survey is needed to know the real incidence of the disease. Pear decline disease is, in many varieties, associated with early leaf reddening although there
Fig. 5 RsaI restriction profiles of a 16S rDNA fragment amplified with U5/P7 from phytoplasmas found in different hosts. Lane 1, Diaphorina putonii; lane 2, plum tree; lane 3, pear tree; lane 4, C. pyri; lane 5, no sample; lane 6, strawberry; lane 7, pepper; lane 8, molecular weight markers.
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Fig. 6 AluI restriction profiles of a 16S rDNA fragment amplified with PD/O1 from phytoplasmas found in pear trees and psyllids from different locations. Lane 1, pear tree from Le´rida; lane 2, pear tree from Sevilla; lane 3, pear tree from Badajoz (although not discernible in the picture); lane 4, C. pyri from Zaragoza; lane 5, C. pyri from Le´rida; lane 6, pear tree from Valencia; lane 7, pear tree from Zaragoza; lane 8, molecular weight markers.
could be varieties such as Limonera where the main symptom is not reddening but weakness of foliage. Symptoms on some trees could be due to several factors other than phytoplasma infection (e.g. waterlogging or a poor rootstock/scion union), illustrating again the lack of reliability of symptoms for diagnosing phytoplasma infection on pears. As indicated by Davies et al. (1992), it is also likely that the uneven distribution of phytoplasmas within
Fig. 7 RsaI restriction profiles of a 16S rDNA fragment amplified with PD/O1 from phytoplasmas found in pear trees and psyllids from different locations. Lane 1, pear tree from Le´rida; lane 2, pear tree from Sevilla; lane 3, pear tree from Badajoz (although not discernible in the picture); lane 4, C. pyri from Zaragoza; lane 5, C. pyri from Le´rida; lane 6, pear tree from Valencia; lane 7, pear tree from Zaragoza; lane 8, molecular weight marker.
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the host plant could have caused some false negative PCR results. PCR positive results in asymptomatic trees could be an indication of the sensitivity of the technique, detecting the pathogen before symptom expression. Some experience of graft transmission of the phytoplasma associated with apricot chlorotic leaf roll revealed that the pathogen can be detected before symptom expression (Avinent, unpublished data). This is one of the most interesting aspects of the application of the technique, since apart from confirming the presence of the pathogen in symptomatic trees some infected but asymptomatic trees could also be removed from nurseries. Although both pairs of primers were used successfully, fPD/rO1 (primer pair for pomefruits) is recommended, because detection with this pair was more sensitive than with fU5/rU3. Although insect transmission has still not been confirmed experimentally, the detection of a phytoplasma in psyllids with the primer pair fPD/ rO1 and the detection of the same RFLP pattern for both pear tree and C. pyri samples are good indications that the phytoplasma in trees and in insects may be the same, and that these insects may be the vectors in Spain, as they are thought to be in other countries (Giunchedi et al., 1994). C. pyri is the most common psyllid in Spanish pear-tree orchards. In recent years populations of C. pyri have increased significantly and the spread of the disease also seems to have increased. Because the disease is difficult to reproduce experimentally with other psyllid species (e.g. C. pyricola) (Davies et al., 1992), new laboratory transmission trials with insects fed on infected trees should be done. This would permit determination of the percentage of infective insects, because not all the insects that feed on infected material are able to transmit the pathogen. It will also be necessary to use pear seedlings as host plants, because C. roseus does not seem to be a good host for pear decline transmission (Davies, personal communication). ACKNOWLEDGEMENTS This work has been funded by the CEE project AIR1-CT92–0659. We thank J. de la Cruz (Plant Protection Service from Badajoz), M. Cambra (Plant Protection Service from Zaragoza), M. P. Baraja (Plant Certification Service from Valencia) and T. Orero (Orero Nurseries from Valencia and Sevilla) for providing material. We also thank S. Oltra and M. A. Martı´nez (IVIA, Valencia) for their help in laboratory.
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