030822

SUMMARY

In spring 2008, in a nursery located in the Como

province (Lombardy, northern Italy) leaf blight symp-

toms were observed on potted box plants (Buxus sem-

pervirens ?Suffruticosa?) followed by a sudden and se-

vere defoliation. Diseased plants, had been imported

from a Belgian nursery. These symptoms were sugges-

tive of box blight, a new disease of the genus Buxus.

The causal agent was isolated and identified through

morphological, cultural and molecular characters as

Cylindrocladium buxicola. C. buxicola was reported as a

pathogen of the genus Buxus in the 1990s in the UK and

New Zealand, and since then has spread throughout

northern Europe. To our knowledge this is the first re-

port of C. buxicola in Italy.

Key words: new disease report, etiology, fungal plant

pathogen, ornamentals.

Box plants of different species are commonly used as

ornamentals in Italy, where they are imported by nurs-

eries as cuttings or potted plants from northern Europe,

particularly Belgium and The Netherlands. In spring

2008, a leaf blight symptom on potted box plants

(Buxus sempervirens ?Suffruticosa?) was observed in a

nursery of the Como province (Lombardy, northern

Italy). Blight was quickly followed by severe defoliation,

which made the plants unmarketable. Diseased propa-

gating material had been imported as symptomless cut-

tings from a Belgian nursery, and arrived in Italy in No-

vember 2006. Symptoms shown by diseased plants were

suggestive of box blight (Henricot et al., 2000), a dis-

ease of Buxus new to Italy.

Box blight, caused by Cylindrocladium buxicola, was

reported from Europe and New Zealand in the 1990s

(Henricot and Culham 2002). The disease was first

found in 1994 in a Hampshire nursery and, by 1997, it

Corresponding author: P. Cortesi

Fax: +39.02.5031678

E-mail: paolo.cortesi@unimi.it

was widespread in the UK (Henricot et al., 2000). In

2000, it was detected in Belgium, first in a private gar-

den, then in other sites throughout the country (Crepel

and Inghelbrecht, 2003). In 2004-2005, a record came

from Germany (Brand, 2005) and in 2007 the disease

was found in a cemetery at Lausanne, Switzerland (Vin-

cent, 2008; www.acw.admin.ch). Box blight causes se-

vere defoliation and dieback of affected plants and, be-

cause of its recent epidemics in northern Europe, in

2007 C. buxicola was placed on the alert list of the Eu-

ropean Plant Protection Organization (www.eppo.org/

quarantine/quarantine.htm), with the goal to hamper its

spreading. To our knowledge, box blight has not yet been re-

ported from Italy, although it occurs in neighbouring

countries (CABI, 2007) and C. buxicola has the poten-

tial to spread long distance through conidia, or through

trading of symptomless infected plants. Assuming that diseased box plants found in Italy

were infected by C. buxicola, the pathogen was isolated

and identified using traditional and molecular methods.

To this aim, diseased B. sempervirens ?Suffruticosa?

plants, were first inspected for symptom observation.

Diseased boxes showed blighted leaves, often aggregat-

ed in variously expanded patches (Fig. 1A). Sympto-

matic leaf area turned light-brown with dark-green bor-

ders, eventually expanding to the petiole and the shoot.

Pathogen sporulation covered the abaxial surface of in-

fected leaves (Fig. 1B). Shoot infections resulted in

dark-brown to black linear or diamond-shaped lesions,

with darker borders, and subsequent blight. New young

shoots eventually developed from healthy twigs parts. Material for scanning electron microscope observa-

tions was processed according to Locci (1972). Briefly,

about 0.5 cm

2of infected leaf area was glued up-side

down to an aluminium microscope stub and exposed to

1% OsO

4vapour overnight. Specimens were then coat-

ed under vacuum with a thin gold film and observed

with a LEO 438VP electron microscope (Leo Electron

Microscopy, UK). For light microscopy, conidia and

conidiophores were transferred from sporulating leaves

to glass slides to be observed under a Orthoplan Leitz

microscope for micro-morphological characters useful

for pathogen identification.

Journal of Plant Pathology (2008), 90 (3), 581-584Edizioni ETS Pisa, 2008 581

SHORT COMMUNICATION

BOX BLIGHT, A NEW DISEASE OF BUXUS IN ITALY CAUSED

BY

CYLINDROCLADIUM BUXICOLA

M. Saracchi, F. Rocchi, C. Pizzatti and P. Cortesi

Dipartimento di Protezione dei Sistemi Agroalimentare e Urbano e Valorizzazione delle Biodiversit? (DiPSA),

Universit? degli Studi, Via Celoria 2, 20133 Milano, Italy

022_JPP382SC_581_COLORE 13-11-2008 16:01 Pagina 581

Results of these observations showed that the

pathogen had cylindrical conidia, rounded at both ends,

straight, hyaline, 1-septate, 42-68 ?m [average 47.3; s.e.

0.8 (N = 20)] x 4-6 ?m [average 3.9; s.e. 0.1 ( N = 20)] in

size. Conidia were formed on conidiophores with peni-

cillate arrangement of fertile branches, and were

arranged in cylindrical clusters held together by colour-

less slime. The stipe of the conidiophores ended with an

ellipsoid vesicle 7-11 ?m [average 9.2; s.e. 0.4 ( N = 8)]

in diameter (Fig. 2). The pathogen was isolated by plating 1 ?l of a conidi-

al suspension (about 10

3 conidia ml -1water) on the sur-

face of potato dextrose agar (PDA) medium in a Petri

dish. After 24 h incubation at 24?C, single hyphae were

collected under a microscope (100x) and transferred to

a fresh plate. Colony morphology and growth of our

reference strain IPV-FW348, were analyzed both on

PDA and on 2% malt extract agar, 2% glucose and

0.1% peptone (MEA). After 7 days growth on MEA

and PDA at 24?C, colonies of strain IPV-FW348 were

approximately 3 and 3.5 cm in diameter, respectively.

Colour of colony reverse on MEA after 7 days at 24?C

was brown in the centre, fading through sienna with a pale luteous halo. The growing mycelium at the margin

was white-cream. No diffusible pigments were ob-

served. Strain IPV-FW348 produced few aerial hyphae.

On PDA the colony reverse, after an incubation of 7

days at 24?C, was characterized by a well defined dark

brown central area surrounded by a cream mycelial

growth. The colony surface was covered by aerial cot-

tony hyphae.

Experimental inoculation tests were carried out with

strain IPV-FW348 grown on PDA for 7 days, by insert-

ing agar plugs, surface down, into wounded twigs of B.

sempervirens ?Suffruticosa?. Wounds were wrapped with

parafilm and the plants were incubated at 24?C for two

weeks. All experimentally infected plants showed blight

symptom on twigs and leaves within two weeks. The

pathogen was reisolated from surface-sterilized wood

chips from symptomatic twigs, thus fulfilling Koch?s

postulates. For molecular analysis, strain IPV-FW348 was grown

on cellophane placed onto the surface of PDA in a Petri

dish. After 7-day incubation at 24?C in the dark, the

mycelium was transferred to an eppendorf tube, frozen

at -25?C, lyophilized, ground to a powder and the DNA

582 Box blight Journal of Plant Pathology (2008), 90(3), 581-584

Fig. 1. A. Buxus sempervirens ?Suffruticosa? infected by C. buxicola showing an expanding area of curled blighted leaves and incip-

ient defoliation. B. Close-up of an infected leaf. The area colonized by\

C. buxicola is light-brown with dark-green borders.

Pathogen propagules cover the abaxial surface.

022_JPP382SC_581_COLORE 13-11-2008 16:02 Pagina 582

extracted using E.Z.N.A. HP Plant DNA Miniprep Kit

(Omega Bio-Tek Inc., USA). The universal primers

ITS1 and ITS4 were used to amplify the ITS-1, 5.8S and

ITS-2 regions of the nuclear rDNA (Whiteet al., 1990).

DNA was amplified in a total of 30 ?l containing 1x

Green Go Taq reaction Buffer (Promega, Italy), 0.1 mM

of each dNTP, 1?M of each primer, 0.9 U of GoTaq

DNA Polymerase (Promega, Italy) and 1.2 ?l DNA.

PCR was performed with a Bio-Rad thermalcycler at the

following conditions: 2 min at 95?C, 25 cycles at 95?C

for 30 sec, 55?C for 30 sec and 72?C for 1 min, and a fi-

nal extension at 72?C for 10 min. PCR products were

checked by electrophoresis in 1.5% agarose gel in Tris-

acetate EDTA buffer stained with ethidium bromide.

The DNA was purified by Wizard SW Gel and PCR

Clean-Up System (Promega, Italy) and sequenced using

the primer ITS1 by Primm (Milano, Italy). The ITS1-

5.8S-ITS2 rDNA sequence was compared with those

available in the EMBL-EBI GeneBank database using

the FASTA program. The 425 bp ITS1-5.8S-ITS2 rD-

NA sequence obtained was identical to the homologous

region of the type strain RHS-9934 of C. buxicola (ac-

cession No. AY078112) (Henricot and Culham, 2002). We have successfully isolated from diseased B. sem-

pervirens ?Suffruticosa? the causal agent of box blight

and fulfilled Koch?s postulates. Scanning electron and

light microscope measurements of conidiophores, coni-

dia and vescicles coupled with morphological characters

of the fungal colony supported the notion that the

pathogen was C. buxicola, a new species recently de-

scribed by Henricot and Culham (2002). In addition,

the ITS1-5.8S-ITS2 rDNA sequence of our reference

strain IPV-FW348 was identical to that of C. buxicola

type strain RHS-9934 determined by Henricot and Cul-

ham (2002). Based on these results, we can conclude that the dis-

ease observed on B. sempervirens ?Suffruticosa? in the

province of Como is box blight caused by C. buxicola

which, to our knowledge, represents the first record of

this disease in Italy. As mentioned above, the Italian nursery imported

symptomless box cuttings in November 2006 and the

first leaf blight symptoms appeared on a few plants in

spring 2007. However, that spring was particularly hot,

so the grower attributed the blight symptom to drought

and destroyed the damaged plants. Spring 2008, howev-

er, was particularly rainy and cool, so the blight was se-

vere and widespread, but only within the same lot of B.

sempervirens ?Suffruticosa ?. In the absence of chemical

treatments, the epidemic caused severe defoliation on

most plants and the nurseryman decided to destroy the

whole lot, which fortunately was small. To date, the

search for box blight on other species of Buxus (B. sem-

pervires, B. sempervires ?Baluer Heinz?B. microphylla

?Winter Gem?, ?Golden Triumph? and ?Rococ??) within

the same nursery has been negative. B. sempervirens ?Suffruticosa? seems to be less tolerant to the disease

than other species within the genus

Buxus(Henricot et

al., 2000), although this claim is based on anecdotal

field observations rather than experimental data, where-

as B. balearica and the genus Sarcococca are the most tol-

erant (Henricot et al., 2008).

In Italy, the genus Buxus is widespread in private gar-

dens and public areas, as well as in nurseries, which

provide planting material often imported from northern

Europe. To date, sound epidemiological data are not

available, but we can infer that the disease spreads long

distance through trade in box plants and locally because

of the abundant production of conidia, when climatic

conditions are favourable and disease control strategies

are absent (at least for Italy). Therefore, the finding of

the disease in our country constitutes a risk for the ex-

tensive use of Buxus which, so far, has not suffered seri-

ous pest and disease damages. Disease management of box blight should be based

on epidemiological knowledge and information on

which fungicides are most effective against the

Journal of Plant Pathology (2008), 90 (3), 581-584 Saracchi et al. 583

Fig. 2.Scanning electron micrograph of C. buxicolaefflores-

cence on the abaxial leaf surface of Buxus sempervirens?Suf-

fruticosa?, showing clusters of conidia (a), conidiophores and

vescicles (b).

022_JPP382SC_581_COLORE 13-11-2008 16:02 Pagina 583

pathogen. Preliminary laboratory essays showed that

only a few fungicides could be operatively effective

(Brand, 2006; Henricotet al., 2008). Therefore, highly

efficient quarantine measures must be implemented to

prevent any further movement of the pathogen with

shipments of box plants.

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584 Box blight Journal of Plant Pathology (2008), 90(3), 581-584

Received July 29, 2008

Accepted September 1, 2008

022_JPP382SC_581_COLORE 13-11-2008 16:02 Pagina 584