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PHIALOPHORA-LIKE FUNGI ASSOCIATED WITH KIWIFRUIT ELEPHANTIASIS A. Prodi, S. Sandalo, S. Tonti, P. Nipoti and A. Pisi Dipartimento di Scienze e Tecnologie Agroambientali, Alma Mater Studiorum, Università degli Studi, Viale Fanin 40, 40127 Bologna, Italy

SUMMARY

An unusual disease, named elephantiasis for its typical symptoms, has been seen in orchards of kiwifruit cv. Hayward in Emilia-Romagna (northern Italy) since 2001. Phialophora-like isolates were obtained from the necrotic wood and were studied in vitro for phenotype and tissue colonization ability. We used primers amplifying the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) for molecular identification. Phaeoacremonium strains were further identified with b-tubulin-specific primers. Strains of Cadophora, Lecythophora and Phaeoacremonium were classified and characterized. The isolates differed in their ability to colonise tissue. P. aleophilum and C. melinii showed the highest colonization index. To our knowledge, this is the first report of Cadophora melinii isolated from kiwifruit plants. Keywords: Actinidia deliciosa, fungal disease, Phaeoacremonium, Cadophora, molecular assays, ITS region.

INTRODUCTION

Kiwifruit (Actinidia deliciosa [(A. Chev.) C.F. Liang et A.R. Ferguson] var. deliciosa) is an important crop of temperate regions. Italy is the leading kiwifruit producer in the world with a total production of 422,335 tons in 2006 (FAO, 2008). In the last fifteen years, A. deliciosa plants with decline symptoms have been observed in several European countries, Chile and New Zealand. In Chile, symptoms of internal wood browning and small and silvery leaves were attributed to Chondrostereum purpureum (Alvarez et al., 1999). Similar wood and leaf symptoms were also seen in several Italian kiwifruit orchards. Symptoms included deterioration of the wood structure and fruits which remained small and did not ripen properly. This syndrome was ascribed to a form of wood decay which can Corresponding author: Paola Nipoti Fax: +39 051 2096722 E-mail: [email protected]

affect 10-year-old plants (Calzarano et al., 1999). Longitudinal trunk sections showed brown areas of hard necrotic tissue later followed by a spongy and friable discolored zone, both symptoms developed from pruning cuts and progressed downwards. Analysis of deteriorated wood revealed the presence of fungi, typically responsible for complex diseases of woody tissue in different plants, e.g. Petri disease of grapevine. As reported by Di Marco et al. (2000) the following fungi were isolated: Phaeoacremonium inflatipes W. Gams, P.W. Crous & M.J. Wingf., P. aleophilum W. Gams, P.W. Crous, M.J. Wingf. & L. Mugnai, P. rubrigenum W. Gams, P.W. Crous & M.J. Wingf., Phaeomoniella chlamydospora (W. Gams, P.W. Crous, M.J. Wingf. & L. Mugnai) P.W. Crous & W. Gams, Phialophora spp., and agents of white decay like Fomitiporia mediterranea M. Fisch. Subsequent investigations, carried out in kiwifruit orchards of Emilia-Romagna (northern Italy), revealed the presence of symptoms differing from normal “decay”. The most obvious was an abnormal increase in trunk diameter (often over 60% at the collar) accompanied by longitudinal bark cracks. In some orchards, the hypertrophy could be present at different heights of the trunk. Because of the lack of decay, this was regarded as a different disease called “elephantiasis” (Nipoti et al., 2003). These symptoms generally occurred in plants more than ten years old, occasionally in 7-year-old ones. Affected plants were distributed at random in the orchard, and disease incidence was estimated to be less than 10% (Nipoti et al., 2006). In cross section a marked brown discoloration of the annual rings was visible, while in longitudinal sections the brown discoloration tended to diminish upwards, though it could extend to the branches. Diseased plants had reduced foliage and bore small unsalable fruits. In Italy, the disease has also been recorded from Piedmont (Mancini and Cotroneo, 1997), Latium (L. Riccioni, personal communication), and Veneto (Nipoti et al., 2007). It occurs also in France (Hennion et al., 2001), northern Spain (Gonzalez Diaz, 2003) and New Zealand (Manning et al., 2003). Due to the importance of the Italian kiwifruit industry, the disease was studied further. In necrotic areas Nipoti et al. (2006) found a complex mycoflora includ-

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ing Fusarium Link ex Fr., Cylindrocarpon Wollenw., Phomopsis (Sacc.) Bubák and phialophora-like fungi (Gams, 2000). Among phialophora-like endophytes, Harrington and McNew (2003) have included some morphologically little-differentiated anamorphs similar to Phaeoacremonium W. Gams, P.W. Crous & M.J. Wingf., Phialophora Medlar, Cadophora Lagerb. & Melin, and Lecythophora Nannf. From kiwifruit plants affected by elephantiasis Hennion et al. (2001) isolated mainly P. aleophilum and P. viticola, whereas Manning et al. (2003) recovered Phialophora alba F.H. Beyma. Recently, Riccioni et al. (2007) have described a new kiwifruit disorder, called “leader die-back”, from A. chinensis var. Hort16A (Zespri Gold) grown in the Latina area (central Italy), characterized by the death of a single cane or the entire leader and by swelling and bark cracking similar to elephantiasis at the infection site. Fungi like Cryptosporiopsis actinidiae P.R. Johnst., M.A. Manning & X. Meier and Cadophora spp were isolated from diseased plants (Riccioni et al., 2007). With our study we investigated the possible involvement of phialophora-like fungi in kiwifruit trunks showing elephantiasis.

MATERIALS AND METHODS

Fungal isolates and culture conditions. From 2001 onwards, in orchards of the Faenza area (Emilia-Romagna) we examined fifty 8- to 25-year-old kiwifruit plants of cv. Hayward showing elephantiasis but no

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macroscopic decay symptoms (Fig. 1A). Fragments of discolored wood (Fig. 1B) were plated in Petri dishes containing potato dextrose agar (PDA 39 g l-1, Difco, USA), amended with streptomycin sulfate (300 mg l-1, Sigma, USA), at 25°C in the dark. Five symptomless plants were also tested as control. In this study, only phialophora-like fungi, among the different fungi isolated, were considered. Seven phialophora-like isolates kindly supplied by Dr. S. Di Marco (CNR, Bologna, Italy) and Dr. J. Dupont (LCP, Laboratoire de Cryptogamie Paris, France), were used as reference isolates (Table 1). Morphological observations. A mycelial block (2–3 mm2), obtained from 14-day-old single conidial cultures of each of the 34 phialophora-like strains, was transferred to Petri dishes and incubated on 2% malt agar (MA, Sigma, USA) at 25, 30 and 35°C in the dark for 14 days. The capacity to grow at 35°C discriminates phialophora-like taxa (Crous et al., 1996; De Hoog et al., 1999; Dupont et al., 2000, 2002). Five plates for each of the 34 strains were prepared for each temperature. This trial was repeated three times. The macroscopic features, i.e., colour of colonies, pigment diffusing into the agar, and microscopic observations, i.e. conidiophore, phialides and conidial morphology, were used to group the phenotypes (Crous et al., 1996; Gams, 2000; Weber, 2002; Mostert et al., 2003, 2006). DNA extraction, PCR amplification, sequencing and phylogenetic analysis. Genomic DNA of phialophoralike strains was extracted from fresh mycelium grown on

Fig. 1. A. Kiwifruit plant affected by elephantiatis, B. Cross section of an infected trunk showing tissue discoloration.

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MA for 14 days, using a protocol by Lohdi et al. (1994). The universal oligonucleotide primers ITS4 and ITS5 (White et al., 1990) were used to amplify part of the nuclear ribosomal DNA (rDNA) by PCR carried out in a final volume of 25 ml containing 5 ml of diluted sample, 0.75 units of GoTaq Flexi DNA Polymerase (Promega, USA), 5x Green GoTaq Flexi Buffer, 0.2 mM dNTP, 3 mM MgCl2 and 2.5 pmol of each primer. Amplifications were performed in a T3 Biometria Thermalcycler, using an initial denaturation step of 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing for 1 min at 56°C and elongation for 1 min at 72°C, with a final extension for 10 min at 72°C. Products were analyzed by electrophoresis in 1% agarose gel in TBE buffer, stained with ethidium bromide (0.4 mg ml-1) and photographed under UV. The molecular weight of the amplified DNA was estimated by comparison with a 100 bp DNA ladder (Promega, USA). The amplified products were purified (Wizard SV Gel and PCR Clean-Up System, Promega, USA) and ligated into the pGEM-T easy vector (Promega, USA), which was then used to transform competent cells of JM109 Escherichia coli and recombinant plasmid DNA from transformed cells was purified (Wizard Plus SV Minipreps DNA Purification System, Promega, USA). Colonies containing the insert were screened by PCR with primers designed on the polylinker of the vector. Sequencing was done by MWG (Germany). Sequences were aligned using the multiple-sequence alignment program Clustal V method (Higgins and

Sharp, 1989) from the Megalign package (DNAStar, USA). Data were analyzed using TREECON software (Van de Peer and De Wachter, 1994), with Jukes and Cantor distance model to obtain a Neighbour-Joining tree constructed using 1000 bootstrap replicates. Bulleromyces albus CBS 6302 sequence (NCBI, AF444663) was used as outgroup. Since ITS data do not sufficiently discriminate all species of Phaeoacremonium, primers amplifying btubulin were used in a touch-down PCR with the reverse primers Pbr6_1, Pbr12, Pbr11 and Pbr8 (Mostert et al., 2006) in combination with the forward universal primers T1 (O’Donnell and Cigelnik, 1997). Colonization and pathogenicity assays. For better and faster growth, each of the 34 phialophora-like isolates were streaked three times in each Petri dish containing MA and maintained at room temperature for 10 days. Three uninoculated MA Petri dishes were used as control. Young sprouts of A. deliciosa cv Hayward, surface-disinfected in 2% vv-1 NaClO for 10 min, were cut into 3 cm portions and laid horizontally over the fungal streaks or placed in uninoculated dishes (Nipoti et al., 1989; Riccioni et al., 2007). After 10 days incubation at room temperature, the sprouts were cut longitudinally. The experiment was replicated four times. The colonization index of each strain was evaluated by the necrosis/length ratio of the sprout segment. Analysis of variance (ANOVA) was performed using Statgraphic Plus 2.1 software. Means were compared according to

Table 1. Reference strains used in this study. Fungus

Strain number

Orign

Host

Phaeoacremonium aleophilum teleomorph: Togninia minima (Tulasne & C. Tulasne) Berlese.

CBS*101006(1)

Italy

A. deliciosa

P. aleophilum

CBS 101008 (1)

Italy

A. deliciosa

P. iranianum

CBS 101357 (1)

Italy

A. deliciosa

P. parasiticum teleomorph: T. parasitica L. Mostert, W. Gams & P.W.Crous

LCP**883537 (2)

USA

Human

P. viticola teleomorph: T. viticola L. Mostert, W. Gams & P.W. Crous

LCP 963886 (2)

France

Vitis vinifera

Cadophora malorum

CBS 101359 (1)

Italy

A. deliciosa

Sweden

Wood chips of Betula verrucosa and B. pubescens

teleomorph: T. minima

Phialophora verrucosa

1

489

CBS 839.69

(2)

Obtained from Dr. S. Di Marco; 2Obtained from Dr. J. Dupont * CBS = Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands ** LCP = Laboratoire de Cryptogamie Paris, France

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Tukey’s test (P = 0.05). Fifty 2-year-old plants of cv. Hayward from a commercial nursery, kept in plastic pots in a greenhouse, were used for pathogenicity assays. A wound, made in the stem 3 cm above soil level, was inoculated with a mycelial agar plug from 2-week-old cultures of strains showing a high colonization ability. To prevent drying, the wounded area was covered with parafilm. Four sets of 10 plants each were inoculated with a mix of Cadophora melinii strains (2, 13, 75, 76), C. luteo-olivacea (20, 61), Lecythophora luteoviridis (26, 64) and P. aleophilum (9, 10, 29, 30, 31). Controls were inoculated with a plug of sterile agar. After 18 months three plants from each set were examined. The stems were cut longitudinally to assess wood discoloration. Fragments taken from 3 cm above the inoculation site were surface-disinfected in 2% vv-1 NaClO for 5 min and plated onto Petri dishes containing MA. The remaining plants were transferred to a field at Zattaglia, near Faenza, to allow development of symptoms.

RESULTS

Fungal isolates and their morphology. Twenty-seven phialophora-like fungi, were isolated and examined microscopically. Twenty-five were identified based on reference strains and the literature (Crous et al., 1996; Gams, 2000; Weber, 2002; Mostert et al., 2003, 2006). Two isolates, 435 and 29/1, were classified by the Centraalbureau voor Schimmelcultures, Utrecht (CBS) as Cadophora melinii (CBS 111743) and Lecythophora luteoviridis, respectively; these were used, along with those supplied by Drs S. Di Marco and J. Dupont, as reference strains. No phialophora-like isolate was obtained from symptomless plants. Observations of 14-day-old single conidial phialophoralike cultures allowed to recognize three phenotypes: A, B and C (Table 2). Phenotype A: Phaeoacremonium spp. (reference strains: CBS 101006; CBS 101008; CBS 101357, LCP 88 3537, LCP 96 3886). The colonies, of varying colour, usually smoke-grey or honey, reached a diameter of 2540 mm in 14 days at 25°C. Some strains produced a yellow pigment diffusible in the agar. Colonies were felty and woolly. Mycelium had branched, septate, hyaline or light brown hyphae occurring singly or in bundles. Conidiophores were short and usually unbranched. The phialides, arising singly from the conidiophores, were subcylindrical or elongate-ampulliform, attenuated at the base, with an inconspicuous funnel-shaped collarette. Conidia, aggregated into round slimy heads at the phialide apices, were hyaline, aseptate, smoothwalled, typically allantoid and sometimes became twoguttulate with age. Eight strains belonged to this pheno-

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type, 75% of them able to grow at 35°C (Table 2). Phenotype B: Cadophora (reference strains: CBS 101359; CBS 111743). The colonies reached a diameter of 38-55 mm in 14 days at 25°C, were predominantly felt with a distinct grey-brown centre, with aggregated hyphae in the centre and radiating hyaline runner hyphae at the edge. The submerged hyphae were branched, hyaline or light brown. Conidiophores could be branched or unbranched, phialides were flaskshaped with a distinct cup-shaped collarette. Conidia were variously shaped ellipsoidal or obovate and heteropolar. Fourteen strains belonged to this phenotype, and did not grow at 35°C (Table 2). Phenotype C: Lecythophora (reference strain: 29/1). The salmon-pink colonies turned brown ageing and reached a diameter of 35-45 mm in 14 days at 25°C. Phialides were single, flask-shaped, with inconspicuous collarettes, often short necks arising as from intercalary cells. Conidia, aggregated in slimy heads, were aseptate, cylindrical to ellipsoidal and contained many small guttules. Five strains belonged to this phenotype and only one strain was able to grow at 35°C (Table 2). None of our isolates belonged to Phialophora sensu stricto. PCR amplifications, sequencing and phylogenetic analysis. The DNA sequence alignment of phialophoralike strains (Fig. 2) supported the division into three distinct phenotypes: Phaeoacremonium spp. (A), Cadophora spp. (B) and Lecythophora spp. (C). The use of b-tubulin primers allowed the identification of six strains of P. aleophilum (548 bp) and two of P. mortoniae (257 bp); none belonged to P. iranianum (545 bp) or P. viticola (542 bp) (Fig. 3). Within Cadophora spp. (phenotype B) seven strains were identified as C. luteo-olivacea, seven as C. melinii and none as C. malorum (Kidd & Beaum) W. Gams (Fig. 2). In phenotype C, five strains of L. luteoviridis were identified (Fig. 2). The sequences of some of our strains are deposited in GenBank (Table 2). Colonization and pathogenicity assays. Blackening in the kiwifruit sprouts was of variable length related to the different phialophora-like strains. No necrosis was visible in the control sprouts. The colonization capacity of each strain was homogeneous in the four replications. The differences were significant (F = 7.44**), and Tukey’s test (P = 0.05) distinguished three groups differing in colonization index (Table 2). P. aleophilum and C. melinii strains reached the highest values (letter c). Eighteen months after inoculation, in the greenhouse trials as well as in the plants transferred to the field, there was no evidence of hypertrophy. In the longitudinally cut stems we observed discolored tissues only in some plants of two sets: P. aleophilum mix and C. melinii mix. Reiso-

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Table 2. Phialophora-like isolates obtained from elephantiasis-affected kiwifruit plants, and some reference strains.

Strain and collection number

NCBI GenBank Emilia-Romagna location Year accession No.

9

DQ404356

Sarna - farm A(*)

2002

Abilty to grow at 35°C

Phenotype

Molecular identification

Colonization Index

Yes

A

P. aleophilum

1.00 c(1)

10

Sarna - farm A

2002

Yes

A

P. aleophilum

1.00 c

11

Sarna - farm A

2002

Yes

A

P. aleophilum

1.00 c

Sarna - farm B

2001

Yes

A

P. aleophilum

1.00 c

Sarna - farm B

2001

Yes

A

P. aleophilum

1.00 c

31

Marzeno

2002

Yes

A

P. aleophilum

0.75 abc

CBS 101006

Reference

Yes

A

P. aleophilum

1.00 c

29 30

69

DQ404355

Fossolo

2003

No

A

P. mortoniae

0.08 a

182

EU427312

Zattaglia

2001

No

A

P. mortoniae

0.12 ab

CBS 101008

Reference

No

A

P. mortoniae

0.07 a

CBS 101357

Reference

Yes

A

P. iranianum

1.00 c

LCP 88 3537

Reference

Yes

A

P. parasiticum

0.08 a

LCP 96 3886

Reference

No

A

P. viticola

0.51 abc

2

Sarna - farm A

2002

No

B

C. melinii

1.00 c

13

Sarna - farm A

2002

No

B

C. melinii

1.00 c

16

Sarna - farm B

2004

No

B

C. melinii

0.75 bc

Modigliana - farm B

2001

No

B

C. melinii

0.70 abc

75

Modigliana - farm A

2003

No

B

C. melinii

1.00 c

76

Modigliana - farm A

2003

No

B

C. melinii

0.98 c

19

DQ404352

435 CBS 111743

DQ404351

Sarna - farm B

2002

No

B

C. melinii

1.00 c

18

DQ404348

Zattaglia

2001

No

B

C. luteo-olivacea

0.07 a

20

DQ404349

Zattaglia

2001

No

B

C. luteo-olivacea

0.61 abc

21

Modigliana - farm B

2001

No

B

C. luteo-olivacea

0.07 a

23

Marzeno

2002

No

B

C. luteo-olivacea

0.46 abc

39

Zattaglia

2002

No

B

C. luteo-olivacea

0.16 ab

40

Modigliana - farm B

2002

No

B

C. luteo-olivacea

0.31 abc

Fossolo

2003

No

B

C. luteo-olivacea

0.42 abc

No

B

C. malorum

0.51 abc

61 CBS 101359

DQ404350

Reference

1

Sarna farm A

2002

Yes

C

L. luteoviridis

0.16 ab

26

Sarna farm A

2001

No

C

L. luteoviridis

0.48 abc

64

Sarna farm A

2002

No

C

L. luteoviridis

0.44 abc

65

Marzeno

2002

No

C

L. luteoviridis

0.15 ab

29/1

Sarna - farm B

2002

No

C

L. luteoviridis

0.20 ab

Yes

D

Phialophora verrucosa

0.19 ab

CBS 839 69

DQ404354

DQ404353

Reference

F = 7.44** 1

*Different farms in the same location. Means of colonization index followed by the same letter are not significantly different (P=0.05) according Tukey’s test.

lations was possible from the discolored wood of all three plants inoculated with P. aleophilum but only from one plant inoculated with C. melinii.

DISCUSSION

Phialophora-like fungi isolated from kiwifruit plants with trunk hypertrophy, comprised members of several

similar genera, which are difficult to distinguish phenotypically (Gams, 2000; Weber, 2002; Harrington and McNew, 2003; Aroca and Raposo, 2007). These fungi occur in many environments such as decaying wood, soil, water and food, and can also cause human mycoses (Schol-Schwarz, 1970; Mostert et al., 2005, 2006). As mentioned, their identification is difficult, especially for Phaeoacremonium, but the monograph of Mostert et al. (2006) has made an important contribution to this aim.

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Fig. 2. Phylogenetic relationships among phialophora-like isolates based on aligned internal transcribed spacer (ITS) sequences and part of 18S rRNA, 5.8S rRNA and 28S sequences. The sequences of C. luteo-olivacea AY249068 and B. albus AF 444663 are from the National Center for Biotechnology Information (NCBI) GenBank. Bootstrap support values (1000 replicates) are shown at the nodes. Scale bar represents a genetic distance of 0.1. B. albus CBS 6302 selected as outgroup.

Phialophora-like fungi from elephantiasis-affected kiwifruit showed variation and comprised at least three phenotypes and five molecular groups. The phenotype A strains were identified as P. aleophilum and P. mortoniae, the former of which showed the highest ability to colonize kiwifruit sprouts, confirming previous findings by Di Marco et al. (2004). Other Phaeoacremonium species have also been isolated from kiwifruit, i.e. P. parasiticum (Di Marco et al., 2004), P. iranianum (Mostert et al., 2006), P. viticola (Hennion et al., 2001) and in our study P. mortoniae, which showed poor colonization ability. The latter fungus was reported by Gramaje et al. (2007) to cause decline of young grapevines in Spain.

Phaeoacremonium species are associated with diseases of various woody hosts, such as grapevine, olive, apricot, date palm, hop bush and oak, either as endophytes or as possible pathogens inducing wilting, dieback or death (Mostert et al., 2005). In particular P. aleophilum is thought to be a precursor of several other pathogens and is considered to be a primary cause of Petri disease and esca, two complex grapevine diseases (Larignon and Dubos, 1997; Mugnai et al., 1999; Khan et al., 2000; Mostert et al., 2006). This role is due to its ability to colonise parenchyma and vascular tissues and to break down polyphenolic compounds (Mugnai et al., 1999). P. aleophilum can also infect kiwifruit propagat-

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ing material (Nipoti et al., 2007) besides grapevine nursery plants (Zanzotto et al., 2007). Fourie and Halleen (2004) emphasized the importance of infected propagation material as a major means of spread of these fungi. The presence of C. melinii has been observed for the first time in kiwifruit. In addition we isolated C. luteoolivacea but not C. malorum. Cadophlora species showed variable ability to colonise kiwifruit sprouts. In the literature some importance has been ascribed to Cadophora species as tissue colonizers. For instance, C. luteo-olivacea was reported as the causal agent of leader die-back of kiwifruit, although this disease is not well studied (Riccioni et al., 2007) and C. malorum, formerly misidentified as Phaeomoniella chlamydospora (reference strain CBS 101359), is thought to be involved in kiwifruit decay (Di Marco et al., 2004). In our study C. melinii was able to colonise tissues better than C. luteoolivacea. Thus it is possible that C. melinii is involved in the aetiology of elephantiasis. All strains of phenotype C belonged to L. luteoviridis. They showed intermediate ability to colonise kiwifruit tissues. So far, Lecythophora spp. have been reported as mainly associated with wood discoloration in Picea abies (Weber, 2002). Fusarium spp., F. solani in particular, and phialophora-like organisms have been isolated with a high frequency in association from elephantiasis-affected plants (Prodi et al., 2006). Since F. solani can cause wood discoloration and incite the development of small swellings in Platanus x acerifolia (Pilotti et al., 2002), it may have a role in the hypertrophy of kiwifruit (Nipoti et al., 2004), thus supporting the suggestion that elephantiasis may be caused by the interaction of different fungi. We have found that P. aleophlium and C. melinii play a pioneering role in disease development due to their ability to colonise kiwifruit tissue, but the subsequent possible interaction with Fusarium spp. needs confirmation. Pathogenicity trials using F. solani in association with different phialophora-like phenotypes, artificially inoculated at different stages, are in progress. The conclusion is that elephantiasis is a disease with a complex etiology, for a better understanding of which the identification of the different fungal species isolated from affected plants represents a preliminary step.

ACKNOWLEDGEMENTS

We thank Dr J. Dupont and Dr S. Di Marco for kindly supplying fungal strains, Dr. R. Credi for his research support, Dr. G. Spada and Dr. S. Graziani for field technical help. This work was supported by Centro Ricerche Produzioni Vegetali (CRPV) (Projects “Nuove fitopatie dell’actinidia” and Fitopatie dell’actinidia: ”carie” ed elefantiasi – Regional Law 28/98).

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Fig. 3. PCR products amplified from genomic DNA of Phaeoacremonium strains using specific primers for b-tubulin of: (i) P. aleophilum (Pbr6_1/T1 – expected product 548 bp): lane 1, strain CBS 101006; lane 2, strain 9; lane 3, strain 10; lane 4, strain 11, lane 5, strain 29; lane 6, strain 30; lane 7, strain 31; (ii) P. iranianum (Pbr12/T1 - expected product 545 bp): lane 8, strain CBS 101357; (iii) P. viticola (Pbr 8/T1 – expected product 542 bp): lane 9, LCP 96 3836; (iv) P. mortoniae (Pbr 11 – expected product 257 bp): lane 10, strain CBS 101008; lane11, strain 69; lane 12, strain 182. Lane M, 100 bp molecular marker.

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Received February 13, 2008 Accepted March 31, 2008

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