144 DISEASE/Botrytis
and A. vitis (biovar 3), regardless of pathogenicity
(Table 2). Finally in 2001 the nomenclature was
again proposed to place all species currently in
the genus Agrobacterium in the closely related genus
Rhizobium.
See also: Breeding: Selection Strategies for Disease and
Pest Resistance. Disease: Bactericides and Fungicides.
Integrated Pest Management.
Further Reading
Binns AN and Thomashow MF (1988) Cell biology of
Agrobacterium infection and transformation of plants.
Annual Review of Microbiology 42: 575ÿ606.
Bouzar H, Jones JB and Bishop JB (1995) Simple cultural
tests for identi®cation of Agrobacterium biovars. In:
Gartland KMA and Davey MR (eds.) Methods in
Molecular Biology, vol. 44, pp. 9ÿ13. Totowa, NJ:
Humana Press.
Bradbury JF (1986) Guide to Plant Pathogenic Bacteria.
Kew, UK: Commonwealth Agricultural Bureaux.
Broembsen S (1994) Extension Facts: Diseases of Roses.
Stillwater, OK: Oklahoma State University.
Dohm A, Ludwig C, Schilling D and Debener T (2001)
Transformation of roses with genes from antifungal
proteins. Acta Horticulturae 547: 27ÿ33.
Gelvin SB (2000) Agrobacterium and plant genes involved
in T-DNA transfer and integration. Annual Review of
Plant Physiology and Plant Molecular Biology 51:
223ÿ256.
Horst RK (1995) Compendium of Rose Diseases, pp.
23ÿ25. St Paul, MN: American Phytopathological
Society.
Kersters K and De Ley J (1984) Genus III. Agrobacterium.
In: Krieg NR and Holt JG (eds.) Bergey's Manual
of Systematic Bacteriology, vol. 1, pp. 244ÿ254.
Baltimore, MD: Williams & Wilkins.
Klee H, Horsch R and Rogers S (1987) Agrobacterium-
mediated plant transformation and its further applica-
tions to plant biology. Annual Review of Plant
Physiology and Plant Molecular Biology 38: 467ÿ486.
Moore LW, Bouzar H and Burr T (2001) Agrobacterium.
In: Schaad NW, Jones JB and Chun W (eds.) Laboratory
Guide for Identi®cation of Plant Pathogenic Bacteria,
3rd edn, pp. 17ÿ35. St Paul, MN: American Phyto-
pathological Society.
Nutter RC, Thomashow MF, Gordon MP and Nester EW
(1981) Agrobacterium: nature's genetic engineer. In:
Panopoulos NJ (ed.) Genetic Engineering in the Plant
Sciences, pp. 17ÿ35. New York: Praeger Press.
Pionnat S, Dessaux Y, Nesme X and Poncet C (1996)
Detection and determination of pathogenic Agrobacter-
ium of roses with PCR. Acta Horticulturae 424:
227ÿ232.
Sheng J and Citovsky V (1996) Agrobacteriumÿplant cell
DNA transport: have virulence proteins, will travel.
Plant Cell 8: 1699ÿ1710.
Botrytis
M L Gleason and S J Helland, Department of Plant
Pathology, Iowa State University, Ames, IA, USA
# 2003, Elsevier Ltd. All Rights Reserved.
Introduction
Grey mould, caused by the fungus Botrytis cinerea
Pers.:Fr., the imperfect stage of Botryotinia
fuckeliana, is among the most damaging diseases of
cultivated roses worldwide, attacking both outdoor
(Figure 1) and greenhouse (Figure 2) plantings as
well as cut roses during storage and transit. Common
names of the disease on roses include botrytis blight
and botrytis rot. The pathogen is ubiquitous in green-
houses, sporulates abundantly on senescing and dead
plant tissues, and is pathogenic on scores of orna-
mental and vegetable crops in addition to roses. Symp-
toms on rose ¯owers include small ¯ecks that develop
into brown, necrotic spots that can ultimately spread
to engulf the entire ¯ower (Figures 3ÿ5). Stem lesions
are elongated, sunken, and black and brown in colour.
High humidity and cool to mild temperatures are con-
ducive to abundant conidia production; free water on
plant surfaces is required for infection. Infections are
frequently latent when roses are harvested, but symp-
toms may develop during storage and shipment. Small,
rounded, black sclerotia are the most common
overseasoning stage of B. cinerea. Valuable cultural
management techniques include: use of partially
resistant cultivars; rigorous sanitation; suppression
of high humidity in greenhouses by forced-air circu-
lation, venting and heating, and in shipment by
adequately ventilated containers; and watering tech-
niques that minimize the time that plants are wet.
Fungicides are widely used to suppress grey mould
of roses, but resistance and other factors can compro-
mise their effectiveness. Numerous experimental
treatments have the potential to supplement or replace
conventional fungicides for grey mould management,
but these treatments are not yet available for
commercial use.
Symptoms
On petals of greenhouse roses, symptoms typically
appear as small ¯ecks that expand into brown,
necrotic spots. These spots can ultimately engulf entire
petals and the receptacle, ®nally causing collapse of
the ¯ower head and abscission of petals. On suscep-
tible roses, one to three lesions per ¯ower are suf®cient
to render a ¯ower unmarketable. Botrytis cinerea
petal infections on greenhouse roses are frequently
 DISEASE/Botrytis 145

Figure 1 Target-like canker that developed on the cane of a

®eld-grown rose after prolonged humid weather. (Courtesy of
RK Jones, North Carolina State University.)

Figure 3 Flower blighted by B. cinerea infection. (Courtesy of

Iowa State University.)

Figure 2 Lesion developing on the petal of a greenhouse-grown

rose. (Courtesy of Iowa State University.)

latent; that is, they are not visible when ¯owers are
harvested, but develop under humid postharvest stor-
age conditions or as ¯owers mature. Severe economic
losses can occur when symptoms appear on ¯owers in
storage or during shipment.
Lesions on ®eld-grown roses, and sometimes those
grown in greenhouses, may develop woolly masses of
tan-brown mycelium and greyish B. cinerea conidia
under humid conditions. Young twigs and cut stubs
die back and large, sunken, elongated, target-like,
black and brown blotches develop on canes. Small,
black, oval to hemispheric sclerotia frequently form
on or just below the cuticle or epidermis in these
lesions, and are ®rmly attached.
Figure 4 Base of ¯ower blighted by B. cinerea infection. (Cour-
tesy of Iowa State University.)
Disease Cycle and Ecology
Botrytis cinerea persists year-round in greenhouses as
conidia, mycelia or sclerotia on living or dead plants,
and as sclerotia or conidia in infested soil. Outdoors,
the fungus overwinters in infested plant debris or soil.
Sclerotia are the most important structures for ®eld
survival, although conidia may also overseason in the
®eld. The survival stages can be spread long distances
146 DISEASE/Botrytis
Figure 5 Blight of entire bloom, caused by advanced B. cinerea
infection. (Courtesy of Iowa State University.)
through movement of infested plant debris or soil.
Sclerotia commonly germinate by producing conidia,
occasionally by producing hyphae, and rarely by pro-
ducing apothecia and ascospores.
Conidia developing from sclerotia, infected plants
or infested plant debris are dispersed in large numbers
by air currents. Sporulation is favoured when air cir-
culation within the plant canopy is reduced or absent.
Botrytis cinerea spores in a greenhouse can origin-
ate within the crop itself or can enter the house
from external sources through vents. Spore germina-
tion and infection are most rapid at temperatures of
22ÿ25  C with free moisture or high relative humidity
(90ÿ100%). Botrytis cinerea is also active at relatively
low temperatures, however, and can cause consider-
able damage to cut-rose ¯owers and plants stored to
0ÿ10  C. When roses in shipping cartons are trans-
ferred to room temperature during transit or at deliv-
ery sites, condensation of water on the cold ¯owers
triggers germination of B. cinerea conidia that were
deposited during the ¯ower production phase,
resulting in lesions.
Although germinating conidia seldom penetrate in-
tact, actively growing tissue, penetration is common
through wounds such as cut stubs of stems and tip-
burned leaves. Ageing ¯ower petals and senescing
leaves are readily colonized. Levels of ethylene, a com-
pound produced during ¯ower senescence, were pos-
itively correlated with grey mould severity in roses.
Susceptibility of roses to grey mould varies with the
growing environment. Susceptibility of a greenhouse-
grown cultivar may differ seasonally; for example,
`Golden Wave' ¯owers grown in a California
greenhouse during winter were more susceptible than
the same cultivar grown in the autumn, presumably
because increased humidity, lower temperatures and
lower light conditions during the winter reduced
cuticle thickness and thus facilitated invasion by
B. cinerea. Seasonal changes in the greenhouse envi-
ronment, including relative humidity and vapour pres-
sure de®cit, can also affect the amount of airborne
conidia and conduciveness to conidial germination
on plant tissues. Greenhouses that did not rigorously
remove senescing and dead plant tissue, on which
B. cinerea can sporulate copiously, were more vulner-
able to severe grey mould outbreaks than greenhouses
with adequate sanitation practices.
Management
Successful management of grey mould requires inte-
gration of the best available strategies, since no single
measure is adequate alone.
Cultural Strategies
Some rose cultivars are moderately resistant to
B. cinerea, but the degree of resistance has not been
clearly determined for most cultivars. In greenhouses
and ®eld plantings, strict sanitation is a critically im-
portant cultural practice. All spent blossoms, fallen
leaves and other plant debris, as well as any symptom-
atic buds, ¯owers and canes, should be removed, from
both plant beds and beneath benches, hauled away
from the vicinity and destroyed. When soil or recycled
potting media are used for pots and plant beds, they
should be steamed at 82  C for 30 min or 71  C for
1 h in order to destroy B. cinerea inoculum.
Greenhouse relative humidity should be held below
85ÿ90% by forced-air circulation, as well as venting
and heating at night. Plant spacing should be adequate
to allow air movement within the crop canopy. The
goal of these practices is to minimize the duration of
periods when moist, still air remains around the plants.
Adequate spacing is also important for outdoor rose
plantings, and selection of sites with adequate
soil and air drainage. Effective weed suppression
also speeds drying and minimizes the duration of wet
periods. Watering at the base of plants is preferable to
overhead irrigation. Where overhead irrigation is un-
avoidable, however, watering during the morning is
advised, since it allows time for the plants to dry before
nightfall.
Since B. cinerea readily invades wounds, it is helpful
to avoid wounding plants unnecessarily.
Improvements in shipping practices can help sup-
press development of grey mould. Using cardboard
boxes perforated with numerous, large ventilation

holes reduced germination of B. cinerea conidia in
comparison to boxes without holes. Promptly unpack-
ing ¯owers and arranging them in vases also sup-
pressed disease by accelerating drying of ¯ower
buds and petals.
Fungicides and Fungicide Alternatives
Fungicides are widely used to suppress B. cinerea in
rose production, both in greenhouses and in the ®eld.
Effectiveness of fungicides can be limited in several
ways, however. First, it is nearly impossible to protect
all ¯owers even when fungicides are sprayed weekly,
since ¯owers that are closed during treatment may
develop and be harvested before the next spray, and
are thus vulnerable to B. cinerea. Second, B. cinerea
has developed widespread resistance to several fun-
gicides that were formerly mainstays of grey mould
control, including those in the benzimidazole and di-
carboximide classes. Third, since the presence of vis-
ible fungicide residues on harvested roses hampers
marketability, many growers prefer to use products
that leave little or no residue.
Several treatments have shown promise as
alternatives or supplements to conventional fungicide
sprays. Dipping of harvested ¯owers into tap water at
50  C for 20ÿ40 s reduced grey mould severity by
60% without reducing vase-life of the ¯owers. Spray-
ing detached rose petals with a solution of GA3, a
gibberellic acid, suppressed development of grey
mould, possibly by delaying petal senescence and
stimulating the formation of endogenous compounds
inhibitory to B. cinerea. Suppression of grey mould
by GA3 was enhanced by addition of the growth reg-
ulator paclobutrazol. However, postharvest spray
applications of GA3 to intact rose ¯owers did not pro-
vide signi®cant suppression of B. cinerea. Grey mould
was reduced on cut roses that had taken up methyl
jasmonate, a naturally occurring compound in many
plants, because methyl jasmonate apparently induced
systemic acquired resistance mechanisms; ¯ower
quality was not impaired by this treatment. Dipping
rose buds in the antibiotic pyrrolnitrin, which is de-
rived from the bacterium Burkholderia cepacia, and
subsequently inoculating with Botrytis cinerea co-
nidia, suppressed grey mould in cold storage as effec-
tively as a conventional fungicide, and also enhanced
poststorage fresh weight gain.
Inhibitors of ethylene can help to delay ¯ower se-
nescence and thus suppress grey mould. Incubating
cut roses in solutions of CaSO4, CaCl2, or sodium
thiosulphate reduced the severity of grey mould symp-
toms by 60%, probably by inhibiting increases in
ethylene production and membrane permeability
associated with natural senescence.
DISEASE/Botrytis 147
The biological control fungus Clonostachys rosea
(Link:Fr.) Schroers, Samuels, Siefert, and W. Gams,
formerly known as Gliocladium roseum, markedly
suppressed sporulation of B. cinerea in rose petals
and leaves when applied before the arrival of
B. cinerea spores, and rapidly colonized dead leaves
and petals as well. This ®nding suggested that C. rosea
has the potential to effectively suppress the production
of B. cinerea conidia in rose production systems.
Although none of these alternative tactics is cur-
rently registered for use against B. cinerea on roses,
they may be useful in the future as supplementary
treatments to decrease reliance on conventional fun-
gicides that are resistance-prone or environmentally
toxic.
See also: Breeding: Selection Strategies for Disease and
Pest Resistance. Disease: Bactericides and Fungicides.
Integrated Pest Management.
Further Reading
Elad Y (1988) Involvement of ethylene in the disease
caused by Botrytis cinerea on rose and carnation ¯owers
and the possibility of control. Annals of Applied
Biology 113: 589ÿ598.
Elad Y (1988) Latent infection of Botrytis cinerea in rose
¯owers and combined chemical and physiological
control of the disease. Crop Protection 7: 361ÿ366.
Elad Y and Volpin H (1991) Heat treatment for the control
of rose and carnation grey mould (Botrytis cinerea).
Plant Pathology 40: 278ÿ286.
Hammer P and Evenson K (1994) Differences between rose
cultivars in susceptibility to infection by Botrytis
cinerea. Phytopathology 84: 1305ÿ1312.
Hammer P and Evensen K (1996) Effects of the production
environment on the susceptibility of rose ¯owers to
postharvest infection by Botrytis cinerea. Journal of the
American Society of Horticultural Science 121:
314ÿ320.
Hazendonk A, ten Hoope M and van der Wurff T (1995)
Method to test rose cultivars on their susceptibility to
Botrytis cinerea during the post-harvest stage. Acta
Horticulturae 405: 39ÿ45.
Horst R (1983) Compendium of Rose Diseases. St Paul,
MN: APS Press, American Phytopathological Society.
Marois J, Redmond J and MacDonald J (1988) Quanti®ca-
tion of the impact of environment on the susceptibility
of Rosa hybrida ¯owers to Botrytis cinerea. Journal of
the American Society of Horticultural Science 113:
842ÿ845.
Meir S, Droby S, Davidson H et al. (1998) Suppression of
botrytis rot in cut rose ¯owers by postharvest applica-
tion of methyl jasmonate. Postharvest Biology and
Technology 13: 235ÿ243.
Morandi A, Sutton J and Maf®a L (2000) Effects of host
and microbial factors on development of Clonostachys
148 DISEASE/Black Spot
rosea and control of Botrytis cinerea in rose. European
Journal of Plant Pathology 106: 439ÿ448.
Nameth S (2002) Botrytis Gray Mold in Greenhouse Floral
Crops. Ohio State University Extension FactSheet
HYG-3070-96.
Rodriguez A, deVis R and Arbelaez G (1999) Effect of a
photoselective plastic and a climatic screen on the
disease caused by Botrytis cinerea pers. in a rose farm.
Acta Horticulturae 482: 227ÿ233.
Shaul O, Elad Y and Zieslin N (1995) Suppression of
botrytis blight in cut rose ¯owers with gibberellic acid:
effects of postharvest timing of the gibberellin treat-
ment, conidial inoculation, and cold storage period.
Postharvest Biology and Technology 6: 331ÿ339.
Volpin H and Elad Y (1991) In¯uence of calcium nutrition
on susceptibility of rose ¯owers to botrytis blight.
Phytopathology 81: 1390ÿ1394.
Black Spot
R Drewes-Alvarez, University of Applied Sciences,
Dresden, Germany
# 2003, Elsevier Ltd. All Rights Reserved.
Black spot is a common disease of roses caused by the
fungus Diplocarpon rosae Wolf (conidial stage:
Marssonina rosae (Lib.) Died.). It causes early defoli-
ation and weakening of plants. All rose varieties are
more or less susceptible to the disease. In this article
facts about the history, the symptoms and the life-
cycle are given. As the fungus is not a strictly bio-
trophe parasite, it can be grown on arti®cial media.
Isolates of the fungus can be differentiated by morpho-
logical traits or according to their interaction with
different roses. Breeding for black spot resistance re-
quires the existence of resistance genes. After evalua-
tion of species and hybrid roses, resistance genes were
found in some species of roses, such as Rosa multi¯ora
Thunb. ex Murray and R. rugosa Thunb. and breeders
are trying to transfer these genes into cultivars by
cross-breeding or to incorporate resistance genes
from other plants into transgenic roses.
History of the Disease
The conidial stage of the fungus was ®rst reported in
1815 in Sweden by Fries. After that there were reports
from Belgium and France and in the second half of the
nineteenth century the disease was also reported from
Germany and the UK. Reports from North America
and Australia followed at the end of the nineteenth
century. The disease was reported from China in 1910
and in 1914 from Japan. More details can be found in
Table 1. All these papers report the fungus in its
conidial stage. The ®rst report of the perfect stage
was by Wolf in 1912. He found apothecia on
overwintered leaves in Ithaca (NY, USA). Nowadays
the fungus is distributed worldwide and is even found
on islands like the Philippines and Hawaii.
Symptoms of the Disease and Lifecycle
of the Fungus
In spring the ®rst infections are caused by spores that
overwintered in fallen leaf-material, or in structures
called acervuli, on stems. Acervuli are fruiting body
structures that the fungus develops in leaves, forming
conidia, initially subarticularly or subepidermally. In
Figure 1 the life cycle of black spot is shown. The
spores stick to the leaf cuticle, germinate and penetrate
into epidermal cells. The fungus forms strands of sub-
cuticular hyphae and grows deeper into the leaf, with
intercellular hyphae sending nutrient-absorbing struc-
tures called haustoria into the plant cells. Small brown
spots appear on the upper surface of the leaves and
develop into irregular spots with fringed margins. In
Figure 2 part of a rose leaf with black spot symptoms is
shown. While the brown spots develop on the leaves,
small black acervuli with new spores (conidia) are
often formed under the cuticle in concentric circles
but are also distributed irregularly. The diameter of
the acervuli can vary from 50 to 400 mm. In Figure 3
a photograph made under an electron microscope
shows the leaf surface with hyphae and three acervuli.
Inside each acervulus there are masses of two-celled
conidia. In Figure 4 another photograph made under
the electron microscope shows a leaf surface with
some ruptured acervuli and surrounding two-celled
conidia. After the rupture of the cuticle, the conidia
are visible as a white, slimy mass in and around the
acervuli (Figure 2). The conidia are spread by rain
Table 1 First reports of black spot in different countries
Country Author and year
Sweden Fries (1815)
Belgium Libert (1826)
France Duby (1830)
Germany Bonorden (1853)
England Berkeley (1860)
Scotland Stevenson (1879)
Italy Saccardo (1884)
USA Scribner (1887)
Australia Cooke (1892)
The Netherlands Oudemans (1904)
Hungary Laszlo (1910)
Data from Aronescu (1934) and Frick (1943).