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Biology and biocontrol potential of

Ampelomyces mycoparasites, natural
antagonists of powdery mildew fungi
a b c a c b
L. Kiss , J.C. Russell , O. Szentiványi , X. Xu & P. Jeffries
a
Plant Protection Institute, Hungarian Academy of Sciences,
H-1525, Budapest, P.O. Box 102, Hungary
b
Research School of Biosciences, University of Kent, Canterbury,
Kent, CT2 7NJ, UK
c
Horticulture Research International, East Malling, Kent, ME19
6BJ, UK

Available online: 19 Oct 2010

To cite this article: L. Kiss, J.C. Russell, O. Szentiványi, X. Xu & P. Jeffries (2004): Biology and
biocontrol potential of Ampelomyces mycoparasites, natural antagonists of powdery mildew fungi,
Biocontrol Science and Technology, 14:7, 635-651

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 Biocontrol Science and Technology (November 2004), Vol. 14, No. 7, 635
/651

REVIEW

Biology and Biocontrol Potential of Ampelomyces

Mycoparasites, Natural Antagonists of Powdery Mildew Fungi

L. KISS1, J. C. RUSSELL2,3, O. SZENTIVÁNYI1, X. XU3

2
AND P. JEFFRIES
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1
Plant Protection Institute, Hungarian Academy of Sciences, H-1525 Budapest,
P.O. Box 102, Hungary; 2Research School of Biosciences, University of Kent,
Canterbury, Kent CT2 7NJ, UK; 3Horticulture Research International, East
Malling, Kent ME19 6BJ, UK

(Received 5 September 2003; returned 4 December 2003; accepted 18 January 2004)

Historically, pycnidial fungi belonging to the genus Ampelomyces were among the first
mycoparasites to be studied in detail and were also the first fungi used as biocontrol agents of
plant parasitic fungi. The interactions between host plants, powdery mildew fungi and
Ampelomyces mycoparasites are one of the most evident cases of tritrophic relationships in
nature although their study has received little attention in fungal and plant ecology so far.
Ampelomyces mycoparasites have now become one of the most advanced in terms of
commercial development of a fungal biocontrol agent, although there is still a need for more
development work to produce a product with reliability approaching that of conventional
chemical treatments. This review summarizes the taxonomy, genetic diversity, life cycle, mode of
action, natural occurrence, host range, biocontrol potential, mass production and commercia-
lization of these mycoparasites and compares the biocontrol ability of Ampelomyces with that
of other fungal antagonists of powdery mildews.

Keywords: Ampelomyces spp., powdery mildew, hyperparasitism, plant protection, integrated

control, biocontrol, fungal antagonists, AQ10, tritrophic interactions

INTRODUCTION
The exploitation of antagonistic trophic interactions between plant inhabiting microorgan-
isms offers an opportunity for their use in biological control of plant diseases, yet few
examples have reached the market. One notable exception is the use of Ampelomyces as a
biocontrol agent for powdery mildew. Powdery mildew infections remain among the most
important plant pathological problems world-wide, both in agriculture and horticulture,

Correspondence to: L. Kiss. Tel: /36-1-4877-566; Fax: /36-1-4877-555; E-mail: lkiss@[Link]

ISSN 0958-3157 (print)/ISSN 1360-0478 (online)/04/070635-17 # 2004 Taylor & Francis Ltd
DOI: 10.1080/09583150410001683600
 636 L. KISS ET AL.

despite extensive research on their pathogenesis, epidemiology and control. These diseases
account for the greatest proportion of the fungicides applied in European agriculture
(Hewitt, 1998). Important crops including wheat (Triticum aestivum ), barley (Hordeum
vulgare ), grape-vine (Vitis vinifera ), apple (Malus domestica ), and several vegetables and
ornamentals, grown in the field or greenhouses, are among the major targets of powdery
mildew fungi. Cultivars resistant or tolerant to powdery mildew infections have been
developed in a number of crops, but their use is limited, especially in fruit and vegetable crops
(Bélanger & Benyagoub, 1997). In other significant crops, such as tomato (Lycopersicon
esculentum ), all the cultivars and hybrids available to the growers world-wide are susceptible
to powdery mildew (Kiss et al ., 2001).
The constraints associated with the use of fungicides and resistant cultivars have led to the
search of alternative methods to control powdery mildews. The efficacy of non-fungicide
products, such as oils, salts, soluble silicon and plant extracts have extensively been studied,
especially in greenhouse production (Bélanger & Benyagoub, 1997; Pasini et al ., 1997;
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McGrath & Shishkoff, 1999). Many potential biocontrol agents of powdery mildews have
also been tested. Among these, the use of mycolytic bacteria (Skidmore, 1976; Highland,
2000) and mycophagous arthropods (Yarwood, 1957; English-Loeb et al ., 1999) have not so
far showed promise of practical control. In contrast, better results have been obtained in
biocontrol trials that have used fungi antagonistic to powdery mildews.
Recently, Kiss (2003) reviewed the ecology, modes of action and biocontrol efficacy of the
approximately 40 fungal species that have been reported as natural antagonists of powdery
mildews or have been tested as potential biocontrol agents. Of these, pycnidial fungi
belonging to the genus Ampelomyces Ces. are the oldest known and the commonest natural
antagonists of powdery mildews that have been intensively studied in crop protection
practice. One Ampelomyces strain has already been formulated, registered and commercia-
lized in some countries by Ecogen, Inc. (Langhorne, PA, USA) as a product under the trade
name AQ10TM Biofungicide. A second biofungicide, Sporodex† , a product containing a
strain of Pseudozyma flocculosa (Traquair, Shaw & Jarvis) Boekhout & Traquair, is now also
available for use against powdery mildews (Paulitz & Bélanger, 2001; Bélanger & Labbé,
2002). AQ10 Biofungicide is one of the few biocontrol products developed against fungal
plant pathogens (Whipps & Lumsden, 2001), with the result that Ampelomyces mycopar-
asites are among the most studied biocontrol agents. In addition, the interactions between
host plants, powdery mildew fungi and Ampelomyces mycoparasites are one of the most
evident cases of tritrophic relationships in nature, because this relationship is common
world-wide and takes place exclusively on aerial plant surfaces, thus facilitating its direct
observation (Kiss, 1998). However, it has received little attention in fungal and plant ecology,
although it could be used as a model to study the significance of mycoparasitism in the
natural dynamics of plant parasitic fungi.
This review is concentrated on the biology and biocontrol potential of Ampelomyces spp.
to provide an insight for future research in fungal and plant ecology as well as in the
development of products for control of plant diseases. It is timely as there is controversy
regarding the identification of pycnidial mycoparasites of powdery mildews (Sullivan &
White, 2000). It should be noted that major changes have recently occurred in the taxonomy
and nomenclature of the Erysiphaceae (Braun et al ., 2002). However, the names of powdery
mildew fungi used in this review are identical with those used in the corresponding references
to avoid confusion. Plant names also follow the matching references.

REVIEW OF LITERATURE
Early Studies of Ampelomyces Mycoparasites
In 1852, a pycnidial fungus associated with powdery mildew colonies covering grape-vine
leaves was described succinctly as Ampelomyces quisqualis Ces. (Cesati, 1852). Since then,
other similar fungi have been reported by a number of mycologists under various names,
 BIOLOGY AND BIOCONTROL POTENTIAL OF AMPELOMYCES 637

such as Byssocystis textilis Riess (Riess, 1852), Cicinobolus florentinus Ehrenb. (Ehrenberg,
1853), Cicinnobolus cesatii (De Bary, 1870), Cicinobolus humuli Faut. (Fautrey, 1890). Some
authors (e.g., Cesati, 1852; Riess, 1852; De Bary, 1870) clearly recognized that these fungi
were parasites of powdery mildews (Figure 1a,b), but others (e.g., von Mohl, 1852; Tulasne,
1856) considered that their pycnidia and conidia were part of the life cycle of the powdery
mildews with which they were found (Figure 2). The first thorough study of these fungi was
carried out by De Bary (1870) who showed that the hyphae of Ampelomyces grow internally
in the mycelia of powdery mildews from cell to cell, and produce their pycnidia in one or two
cells of the hyphae, conidiophores (Figure 1b), conidium initials and ascocarps of their
fungal hosts. De Bary (1870) proved experimentally that the intracellular hyphae can grow
out from the parasitized cells when placed in water for a few hours (Figure 1a). In addition,
De Bary (1870) carried out cross-inoculation experiments demonstrating that an Ampelo-
myces mycoparasite collected from a given powdery mildew species could also produce
intracellular pycnidia in mycelia of other powdery mildew species. His pioneering work was
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the first detailed study published of an interfungal parasitic relationship. Emmons (1930)
subsequently published an extensive cytological study describing in detail the penetration,
growth and sporulation of Ampelomyces in the ascocarps of powdery mildews. The potential
for biocontrol was realized around the same time and Yarwood (1932) reported the
treatment of powdery mildew infected plants with a conidial suspension of Ampelomyces,
thus carrying out the first biocontrol experiment against a plant pathogenic fungus. Other
early literature was mainly observational (e.g., Fautrey, 1890; Griffiths, 1899; Hino and Kato,
1929; Clare, 1964; Da Ponte, 1966), and reported only the occurrence of Ampelomyces in
various powdery mildews rather than adding to the knowledge regarding biology, physiology
or biocontrol potential.

Taxonomy, Nomenclature and Genetic Diversity of Pycnidial Mycoparasites of Powdery

Mildews
Taxonomic controversies appeared soon after the genus Ampelomyces was erected by Cesati
(1852). The generic name Cicinnobolus was commonly used until the 1970s (Donk, 1966)
despite the priority of the name Ampelomyces being noted by Rogers (1959) and Speer
(1978a). In addition, some authors split the genus in more than 40 taxa describing new
species, formae and new combinations (comb. nov.) based mainly on the host plants or host
powdery mildews in which the mycoparasites were found (e.g., Da Ponte, 1966; Rudakov,

FIGURE 1. De Bary’s drawings from 1870. (a) An intracellular hypha of Ampelomyces growing out from the
powdery mildew mycelium. (b) An intracellular pycnidium of Ampelomyces in a powdery
mildew conidiophore.
 638 L. KISS ET AL.
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FIGURE 2. Tulasne’s drawings redrawn by Moesz (1912) showing the pycnidia of Ampelomyces as one of
the fruiting bodies of powdery mildews.

1979). The descriptions of these species and other taxa are formally still valid, although their
names are not currently in use as there is no evidence for host specificity in Ampelomyces (De
Bary, 1870; Blumer, 1933, 1967; Philipp & Crüger, 1979; Kranz, 1981; Sztejnberg et al ., 1989;
Kiss & Vajna, 1995). Consequently, current practice is to consider that the genus is
monospecific, and that A. quisqualis is a common pycnidial intracellular mycoparasite of all
powdery mildews world-wide. However, in the absence of a formal taxonomic reassessment
of the many validly described species and other taxa, the use of the binomial ‘A. quisqualis ’
for all the pycnidial hyperparasites of powdery mildew fungi is not technically correct and a
taxonomic revision is clearly needed (Sutton, 1980; Kranz, 1981).
This situation is further complicated by a variation in the morphology and cultural
patterns of different ‘Ampelomyces ’ isolates, and this has suggested that the binomial ‘A.
quisqualis ’ has been applied to a range of isolates representing a species complex (Kiss &
Vajna, 1995). However, in the absence of detailed studies, these fungi will still be described
under the ‘Ampelomyces ’ epithet (Kiss, 1997a). Differences were reported in isolates of
pycnidial mycoparasites of powdery mildews in the morphology of conidia (Speer, 1978b;
Rudakov, 1979), pycnidia (Clare, 1964; Belsare et al ., 1980; Shin, 1994), cultural
characteristics (Yarwood, 1932; Mhaskar, 1974; Mhaskar & Rao, 1974; Rudakov, 1979;
Sz. Nagy & Vajna, 1990; Kiss, 1997a), and rDNA ITS sequences (Kiss, 1997a; Kiss &
Nakasone, 1998; Sullivan & White, 2000). Based on the growth rate of these isolates in
culture, two types of growth can be distinguished easily under various cultural conditions.
Firstly, there are fast growing isolates which extend at 3
/4 mm radial growth/day1 at room
temperature. Secondly, there are slower-growing isolates with a radial growth rate of 0.1
/1
mm day 1 (Mhaskar, 1974; Rudakov, 1979; Sz. Nagy & Vajna, 1990; Kiss & Vajna, 1995).
An RFLP analysis showed high genetic diversity in the rDNA ITS region of 46 Ampelomyces
isolates obtained from different parts of the world (Kiss, 1997a). Fast growing and slow
growing isolates were distinguished by different RFLP patterns. A molecular phylogenetic
study of rDNA ITS sequences obtained from ten isolates supported the RFLP analysis, and
revealed that slow growing and fast growing isolates were separated into two distinct clades
which may not even be congeneric (Kiss & Nakasone, 1998). Slow growing isolates were
genetically more diverse, while fast growing ones showed a close phylogenetic relationship
with Epicoccum nigrum (Kiss & Nakasone, 1998) which is known to have a pycnidial,
Phoma -like state (Arenal et al ., 2000).
Recently, Sullivan & White (2000) suggested the possibility that at least some of the fast
growing ‘Ampelomyces ’ isolates were confused with Phoma glomerata (Cda) Wollenw.
isolates. These authors found that fast growing isolates typically came from sessile pycnidia
 BIOLOGY AND BIOCONTROL POTENTIAL OF AMPELOMYCES 639

found on mildew-infected leaves, whilst the slow growing isolates came from intracellular
pycnidia. ITS1 sequence analysis demonstrated that the fast growing isolates clustered in a
clade typified by Phoma species, whilst the slow growing isolates were distinct (Sullivan &
White, 2000). This study supported the results obtained by Kiss and Nakasone (1998). It
seems likely that the fast growing isolates are, in fact, Phoma species, whilst the true
Ampelomyces isolates are always slow growing in culture and always produce intracellular
pycnidia in powdery mildew mycelia. A more thorough taxonomic investigation is
warranted, as some previous authors have identified fast growing isolates as Ampelomyces
spp. (e.g., Linnemann, 1968; Mhaskar, 1974; Rudakov, 1979; Sz. Nagy & Vajna, 1990; Kiss,
1997a). Ampelomyces quercinus, A. humuli and A. heraclei , for example, are species names
based on fast growing isolates (Rudakov, 1979), but these fungi fall within the Phoma/
Epicoccum clade on the basis of phylogenetic analyses (Kiss & Nakasone, 1998; Sullivan &
White, 2000).
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Life Cycle
Conidia of true Ampelomyces isolates are produced in pycnidia developed intracellularly in
the mycelia of powdery mildew fungi (Figures 1b and 2). Conidia are unicellular, hyaline,
mostly guttulate and are embedded in a mucilaginous matrix inside the pycnidia. In the
presence of water, these matrices swell and conidia are released from intracellular pycnidia as
a cirrhus by the rupture of the pycnidial wall. In ca . 10
/20 h under conditions of high
humidity, conidia germinate and the hyphae of the mycoparasites can then penetrate the
hyphae of powdery mildews in their vicinity. The concentration of Ampelomyces conidia on
the leaves is also relevant as Gu and Ko (1997) showed that germination rapidly decreases
above a concentration of 106cfu ml1 due to the production of a self inhibitor. After
penetration, the hyphae of the mycoparasite continue their growth internally and produce
their intracellular pycnidia after 5
/8 days in the mycelia of their host fungi (Figure 1b). A
high RH of the environment enhances the internal growth and sporulation of Ampelomyces
(Jarvis & Slingsby, 1977; Philipp & Crüger, 1979).
Conidia can be dispersed within the plant canopy by rain-splash or water run off from
plant surfaces. Ampelomyces can also spread to long distances as hyphal fragments in
parasitized and detached powdery mildew conidia (Jarvis & Slingsby, 1977; Speer, 1978b;
Sundheim, 1982; Philipp et al ., 1984). When these parasitized air-borne conidia land close to
any powdery mildew colony under humid conditions, the outgrowing hyphae of Ampelo-
myces can penetrate their mycelia. Thus, Ampelomyces can be transported far from its
original fungal host and can parasitize other powdery mildew species. After penetration, the
hyphae of Ampelomyces invade the host mycelia internally, and produce their pycnidia
mostly in the conidiophores and young, immature ascocarps of powdery mildews.
Occasionally, they also produce pycnidia in the invaded hyphal cells. The life cycle starts
again when pycnidia are mature. Emmons (1930) presented equivocal observations regarding
a putative teleomorph, but this was not subsequently reported in detail, and there is no other
record of a sexual stage in Ampelomyces.
Until recently, little was known about the overwintering of Ampelomyces. De Bary (1870),
Emmons (1930) and Yarwood (1939) found that the fungus can produce pycnidia
saprophytically in the senescent or dead plant tissues at the end of the season, and suggested
that these structures served as overwintering structures for Ampelomyces in the field.
However, Falk et al. (1995a,b) considered that saprophytic pycnidia produced in leaf debris
were not significant for the overwinter survival of Ampelomyces in North American
vineyards. They showed that pycnidia of Ampelomyces survived until the next season mainly
in the parasitized ascocarps of Uncinula necator produced on the bark of grapevine stocks.
Similarly, Marboutie et al. (1995) found that pycnidia of Ampelomyces overwintered in the
parasitized ascocarps of Sphaerotheca pannosa on the bark of peach trees. Conidia of
Ampelomyces are probably rain dispersed from the parasitized ascocarps in spring, and then
 640 L. KISS ET AL.

germinate on the young leaves and, if powdery mildew colonies are present, penetrate their
hyphae and invade their mycelia (Falk et al ., 1995a).
Recently, a detailed study of the overwintering of Ampelomyces on apple trees and 13
other woody and herbaceous host plant species revealed that these mycoparasites can survive
the winter in many different ways on the host plant surfaces. In vitro experiments
demonstrated that the overwintered pycnidia of Ampelomyces collected from the host
plants in the spring, and produced in both the conidiophores and the immature ascomata of
powdery mildews during the previous season, can initiate the life cycle of these
mycoparasites (Szentiványi & Kiss, 2003). Both conidia found in some of the overwintered
pycnidia and cells of the pycnidial walls of empty pycnidia germinated in the spring and gave
rise to new intracellular pycnidia of Ampelomyces when powdery mildew colonies were
inoculated with them in vitro. Similar experiments showed that the thick-walled, brownish
resting hyphae of Ampelomyces, described for the first time by De Bary (1870), can also serve
as sources of primary inocula of Ampelomyces in the spring (Szentiványi & Kiss, 2003). So,
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this study demonstrated that the conidia, the pycnidial cells and the cells of the resting
hyphae of Ampelomyces produced in the mycelia of powdery mildews during the previous
season can all initiate the life cycle of these mycoparasites in the spring.

Mode of Action
Some biocontrol fungi have more than one mechanism to antagonize their hosts (Jeffries &
Young, 1994). Trichoderma spp., for example, produce antifungal compounds, act as
mycoparasites, induce plant defence mechanisms and can stimulate plant growth (Howell,
2003). In contrast, Ampelomyces acts directly by invasion and destruction of host cytoplasm.
Ampelomyces kills the parasitized powdery mildew cells by causing a rapid degeneration of
the cytoplasm (Hashioka & Nakai, 1980). These mycoparasites suppress both asexual and
sexual sporulation of the attacked powdery mildew mycelia by colonizing and destroying the
conidiophores, and the immature ascocarps, respectively. The early stage of mycoparasitism
is apparently biotrophic, but the invaded cytoplasm then begins to die and a necrotrophic
interaction results (Hashioka & Nakai, 1980; Sundheim & Krekling, 1982). Philipp et al.
(1984) noted that the parasitized powdery mildew colonies can continue their radial growth,
but their sporulation stopped soon after Ampelomyces penetrated their mycelia. Similarly,
Shishkoff and McGrath (2002) showed that Ampelomyces could not stop the spread of
powdery mildew colonies in vitro but did reduce the amount of inoculum produced by each
colony. Toxin production has not been detected in Ampelomyces (Beuther et al ., 1981) in
contrast to other pycnidial mycoparasites, such as Coniothyrium minitans Campbell
(Machida et al ., 2001; McQuilken et al ., 2002, 2003).
Mycoparasitic attack of powdery mildew mycelia by Ampelomyces results in a reduction in
the negative effects of the pathogen on the host plant. Abo-Foul et al . (1996) showed that
infected cucumber plants regained vigour after being treated with Ampelomyces which killed
the pathogen. Eight days after treatment with a conidial suspension of Ampelomyces,
chlorophyll content and also CO2-fixation in the infected cucumber leaves were almost the
same as in uninfected controls. These results were recently supported by Romero et al.
(2003), who found that a treatment with Ampelomyces significantly increased the chlorophyll
content of detached and mildew-infected melon leaves maintained in vitro.
The presence of host fungi is recognized by Ampelomyces, and a water-soluble substance
from powdery mildew conidia was shown to stimulate the germination of Ampelomyces
conidia in vitro (Gu & Ko, 1997). Directed growth of germ-tubes of Ampelomyces towards
powdery mildew hyphae has also been observed (Sundheim & Krekling, 1982). As with
phytopathogenic fungi, penetration of the host cell wall is likely to involve both enzymatic
and mechanical processes. Appressorium-like structures were reported at the point of
penetration (Sundheim & Krekling, 1982). Extracellular lytic enzymes have been identified in
liquid cultures of Ampelomyces, which may play a role in the degradation of the powdery
 BIOLOGY AND BIOCONTROL POTENTIAL OF AMPELOMYCES 641

mildew hyphal walls during penetration (Philipp, 1985). Rotem et al. (1999) demonstrated
that an exo -b-1,3-glucanase is excreted both in culture and during mycoparasitism, and
showed that culture filtrates of an Ampelomyces isolate could cause degradation of hyphal
walls of Sphaerotheca fusca in the absence of active mycelium.

Ecological Aspects of the Mode of Action of Ampelomyces

In general, when mycoparasites are used as biocontrol agents, a certain level of disease has to
be tolerated as they can only attack established infections (Fokkema, 1993). Microbial
antagonists which act through antibiosis against powdery mildews, such as Pseudozyma spp.
or Tilletiopsis spp., can kill powdery mildew colonies rapidly, causing complete plasmolysis
of their cells (Bélanger & Labbé, 2002). As Ampelomyces spp. act against powdery mildews
through mycoparasitism, without producing antifungal compounds, they destroy the
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powdery mildew colonies only slowly, taking 5
/7 days. In order to maintain control, the
growth and spread of their fungal hosts must be slower than that of Ampelomyces. The
sporulation rate of powdery mildew colonies depends on the inoculum density, physiological
patterns of the host plant, abiotic factors, and so on (Yarwood, 1957; Rouse et al ., 1984;
Bushnell, 2002). When the sporulation and spread of these pathogens is intense,
Ampelomyces mycoparasites applied as biocontrol agents cannot control them completely.
In these cases, they lag behind the spread of the disease, reducing its severity and limiting its
negative effect on the infected plants.
The high RH requirement of Ampelomyces (Jarvis & Slingsby, 1977; Philipp & Crüger,
1979; Sundheim, 1982; Falk et al ., 1995a,b) represents another limiting factor in its use in
biocontrol (Verhaar et al ., 1999; Paulitz & Bélanger, 2001). The dry conditions that usually
favour the development of powdery mildews are not conducive to the development of
Ampelomyces.
As powdery mildews are biotrophic pathogens infecting the aerial parts of their host
plants, potential biocontrol agents need to be active in the phyllosphere, interacting with the
living host plant tissues in a tritrophic system (Kiss, 2001). In general, biocontrol trials of
phyllosphere plant pathogenic fungi have been less successful than those against rhizosphere
pathogens. The phyllosphere is a much more dynamic environment as the leaves have a
limited lifetime compared to roots (Fokkema & Schippers, 1986; Andrews 1990, 1992). Thus,
mycoparasites have a shorter window of attack.
Under natural circumstances, A. quisqualis may not be observed until late in the growing
season on many crops, long after powdery mildews have infected the aerial parts of their host
plants (Gadoury & Pearson, 1988; Rankovic, 1997; Kiss, 1998). Thus, powdery mildew
epidemics usually reach damaging levels before their growth and sporulation are arrested by
A. quisqualis (Gadoury & Pearson, 1988; Falk et al ., 1995a,b). Since the early dispersal and
establishment of the mycoparasite onto the first formed mildew colonies is the most
important factor in the degree of suppression of powdery mildew on leaves and fruit (Falk et
al ., 1995b), application must be targeted to ensure earlier infection of the powdery mildew
pathogens. However, it should be remembered that, by definition, biotrophic pathogens
sporulate on living host tissue. This implies that some level of disease must be tolerated when
mycoparasites are used for control (Fokkema, 1993).
The only powdery mildew structures that are exposed for a longer period of time to
mycoparasitic attack are the overwintering ascomata on the bark of woody crops such as
grape-vine. However, biocontrol of grape-vine mildew through mycoparasitism by Ampelo-
myces was not effective (Falk et al ., 1995a). In conclusion, from an ecological perspective,
there are a number of biotic and abiotic factors that do not seem favourable for the activity of
Ampelomyces against powdery mildew fungi, yet the widespread, natural occurrence of these
fungi would suggest otherwise.
 642 L. KISS ET AL.

Natural Occurrence and Host Range of Ampelomyces

Ampelomyces spp. have been reported from over 65 species (eight genera) of the
Erysiphaceae from across the world (e.g., Hino & Kato, 1929; Emmons, 1930; Blumer,
1933; Clare, 1964; Belsare et al ., 1980; Hanlin & Tortolero, 1984; Hijwegen & Buchenauer,
1984; Sz. Nagy & Vajna, 1990; Puzanova, 1991; Tsay & Tung, 1991; Paulech et al ., 1993;
Shin, 1994, Falk et al ., 1995a; Kiss 1997b; Rankovic, 1997, Kiss 1998). A 4-year study of
both the natural incidence of Ampelomyces and the intensity of mycoparasitism in the field
in a total of 27 species (nine genera) of powdery mildew fungi infecting 41 host plant genera
showed that, in seven out of the nine powdery mildew genera studied, pycnidia of
Ampelomyces were present in approximately 15
/42% of samples (Kiss, 1998). However,
they parasitized only approximately 1
/15% of the total of the host fungal mycelia in these
seven genera of the Erysiphaceae. In contrast, the natural mycoparasitism of the host
mycelia was extremely high (
/65%) in another powdery mildew genus, Arthrocladiella ,
represented by a single species, A. mougeotii , infecting Lycium plants (Kiss, 1998). Other
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field surveys also noted the widespread occurrence of Ampelomyces in mycelia of A.

mougeotii on Lycium (e.g., De Bary, 1870; Rankovic, 1997). Surprisingly, Ampelomyces was
rarely found in Blumeria graminis (DC.) Speer (Kiss, 1997b), the only powdery mildew
fungus that infects monocotyledonous plants, although in vitro inoculation of B. graminis
with Ampelomyces was as successful as it was with other species of the Erysiphaceae infecting
dicotyledonous plants (Sztejnberg et al ., 1989; Kiss & Vajna, 1995; Mairovich et al ., 1996).
In vitro, the germination of Ampelomyces conidia was equally stimulated by the presence of
conidia of A. mougeotii and B. graminis (Gu & Ko, 1997), although, in the field, these two
species were the most and the less parasitized fungal hosts among the 27 powdery mildew
species examined (Kiss, 1998). So, no correlation was found between the field data and the in
vitro mycoparasitic activity of Ampelomyces.
A number of other putative hosts of Ampelomyces reported in the older literature should
also be mentioned here. Naumoff (1913) described and illustrated an Ampelomyces species
producing internal pycnidia in the mycelia of Bremia lactucae Regel, the causal agent of
lettuce downy mildew. However, Blumer (1933) failed to infect lettuce downy mildew with
Ampelomyces when tried to repeat these findings. Other pycnidial fungi, identified as
Ampelomyces quisqualis, were reported as mycoparasites of a number of species of the
Mucorales (Linnemann, 1968). Similarly, a pycnidial fungus found in the hyphae of Fusarium
lateritium Nees was described as an Ampelomyces species (Teterevnicova-Babajan & Nelen,
1975). These records have not been confirmed since and should be regarded as doubtful.
They may represent observations made on Phoma -like fast growing pycnidial fungi referred
to earlier in this review.

Biocontrol Potential
Yarwood (1932) was first to show the potential of Ampelomyces as a biocontrol agent to
control clover mildew (Erysiphe polygoni DC) in a basic experiment, which reproduced the
results of a natural epidemic recorded in the previous year. However, as with many
biocontrol agents, little other work was done as fungicides were effective and were the
accepted method for mildew control in most crops world-wide. It was not until the 1970s that
interest increased in biocontrol, as chemical controls began to break down through the
emergence of fungicide resistance. It is now becoming increasingly clear that chemical control
is not always a sustainable approach, and there is world-wide concern about possible
environmental drawbacks of fungicides (Fokkema, 1993). In addition, the biological control
of insects, which has now become common practice in glasshouse crops, may be adversely
affected by fungicide use (Dik et al ., 1998). As a consequence, more recent research has
focused on the development of other, non-chemical, means of managing powdery mildew
which can be used either in organic production or in an integrated pest management
programme.
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TABLE 1. A list of powdery mildew fungi and their host plants on which the biocontrol potential of different Ampelomyces isolates has been assessed

Crop Powdery mildew fungus References

BIOLOGY AND BIOCONTROL POTENTIAL OF AMPELOMYCES

Apple Podosphaera leucotricha Odintsova, 1975; Sztejnberg, 1979; Bosshard et al ., 1987
Black currant Sphaerotheca mors-uvae Davydova et al ., 1985
Beetroot Erysiphe communis Puzanova, 1984
Carrot E. umbelliferarum, Oidium sp. Sztejnberg, 1979; Sztejnberg & Mazar, 1985; Sztejnberg et al ., 1989
Cucurbits Sphaerotheca spp. Jarvis & Slingsby, 1977; Philipp & Crüger, 1979; Sztejnberg, 1979; Hartmann et al ., 1984;
Philipp et al ., 1982, 1984, 1990; Puzanova, 1984; Sztejnberg & Mazar, 1985; Philipp & Hellstern, 1986;
Diop-Bruckler & Molot, 1987; Hijwegen, 1988, 1989; Sztejnberg et al., 1989; Sundheim, 1982;
Sundheim & Amundsen, 1982; Epton & Hamed El Nil, 1993; Shin & Kyeung, 1994;
McGrath & Shishkoff, 1999; Dik et al ., 1998; Elad et al ., 1998; Lee, 1999
Grape-vine Uncinula necator Puzanova, 1991; Hofstein & Fridlender, 1994;
Yakushkina et al ., 1994; Whitesides, 1994; Falk et al ., 1995a,b; Daoust & Hofstein, 1996; Monchiero et al ., 1996
Mango O. mangiferae Sztejnberg et al ., 1989
Mulberry Phyllactinia suffulta Sztejnberg, 979
Peach S. pannosa var. persicae Marboutie et al ., 1993, 1995
Pepper Leveillula taurica Sztejnberg, 1979; Diop-Bruckler & Molot, 1987
Red clover E. polygoni, E. communis Yarwood, 1932; Puzanova, 1984
Rose S. pannosa var. rosae Puzanova, 1984; Whitesides, 1994; Pasini et al ., 1997; Verhaar et al ., 1999
Strawberry S. macularis f. sp. fragariae Nelson & Gubler, 1995
Sugar-beet E. betae Sztejnberg, 1979
Zinnia sp. Oidium sp. Sztejnberg, 1979

643
 644 L. KISS ET AL.

The first significant trial using Ampelomyces was reported by Jarvis and Slingsby (1977)
who used conidial suspensions of the mycoparasite to control cucumber powdery mildew in
greenhouse trials successfully. The control was enhanced when interspersed with water
sprays. Since this work, there have been many other positive examples where Ampelomyces
has been used to control a range of powdery mildews on several crops (Table 1), and these
have paved the way for commercialisation (Hofstein & Fridlender, 1994; Daoust & Hofstein,
1996; Hofstein et al ., 1996). This was also supported by the lack of any acute oral toxicity of
Ampelomyces cultures in rats. A standard eye irritation test with Ampelomyces in rabbits was
also negative, suggesting that a fungal biocontrol product based on these mycoparasites can
be used without any human health hazards in the plant protection practice (Sundheim &
Tronsmo, 1988).
The most successful biocontrol experiments using Ampelomyces were carried out in
greenhouses where the RH was kept high (e.g., Jarvis & Slingsby, 1977; Sundheim, 1982;
Philipp et al ., 1990), or in the field where free water was frequently available on the treated
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leaves (e.g., Sztejnberg et al ., 1989; Falk et al ., 1995b). However, the efficacy of Ampelomyces
always decreased rapidly when the RH was below 90
/95% (e.g., Philipp et al ., 1984; Philipp
& Hellstern, 1986; Verhaar et al ., 1999). Clearly, the high RH requirement of Ampelomyces
represents one of the major obstacles in its use as a reliable biocontrol agent.
Some attempts were made to enhance the efficacy of Ampelomyces at lower RHs. Epton &
Hamed El Nil (1993) selected isolates which were able to germinate at a higher vapour deficit
than the wild types. A number of additives, such as an emulsion of 1% paraffin oil (Philipp &
Hellstern, 1986) or a 0.3% mineral oil surfactant (Hofstein et al ., 1996), were also reported to
increase the biocontrol performance of Ampelomyces at lower RHs. However, some of these
additives can control powdery mildews directly (Verhaar et al ., 1996; Pasini et al ., 1997).
Shishkoff and McGrath (2002) found that Add-Q, a spray adjuvant recommended to be used
together with AQ10 Biofungicide, was as effective alone against cucumber powdery mildew
as combined with AQ10. Therefore, the effect of the additives and that of Ampelomyces
should be clearly distinguished when assessing the efficacy of a biocontrol experiment.
Recently, some trials have been reported where the treatments with Ampelomyces were
ineffective, and suggested that only very limited control of powdery mildew could be
expected in the plant protection practice (Dik et al ., 1998; McGrath & Shishkoff, 1999;
Verhaar et al ., 1999; Shishkoff & McGrath, 2002).
In most experiments, Ampelomyces was sprayed onto infected plants as a conidial
suspension and the applications were repeated several times during the season to ensure a
high level of control (e.g., Sztejnberg et al ., 1989; Philipp et al ., 1990). In order to reduce the
number of applications in a vineyard, Falk et al . (1995b) devised a method to culture
Ampelomyces on cotton wicks. The inoculated wicks were suspended in grape-vine trellises to
allow natural dispersal of the mycoparasite in rain. This method reduced the number of
applications required, but depended on the weather conditions in the vineyard. As the
artificially released mycoparasites, together with the naturally occurring ones, destroyed a
part of ascomata of grape-vine powdery mildew in autumn, which ensure the perpetuation of
the disease in New York vineyards (Gadoury & Pearson, 1988), they contributed to a
significant delay in the powdery mildew epidemic in the next season (Falk et al ., 1995a,b).
Thus, an additional benefit of this biocontrol experiment was the reduction of the
overwintering inocula of grape-vine powdery mildew.

Mass Production and Commercialization

The scale-up of Ampelomyces inoculum for biocontrol purposes was one of the crucial steps
towards its commercialization and practical use in plant protection. Sztejnberg et al . (1990)
developed and patented (European Patent Office, publ. no. 0353662/1988) a simple and
inexpensive method for production of large amounts of Ampelomyces conidia in fermentors.
Based on this method, different formulations of Ampelomyces were tested in various crops,
 BIOLOGY AND BIOCONTROL POTENTIAL OF AMPELOMYCES 645

especially in grape-vine. The early attempts to introduce a fungal biocontrol product into the
grape-vine industry failed because of its poor formulation and low efficacy (Hofstein et al .,
1996). Finally, an improved product, AQ10TM Biofungicide, produced by Ecogen, Inc. (USA)
obtained EPA registration in 1995 for use in the control of grape-vine powdery mildew. It is
available as a conidial preparation dry-formulated in a stable water-dispersible granule
(Daoust & Hofstein, 1996). Demonstration trials showed that spraying with AQ10 was as
effective as conventional fungicides, providing that the treatment (at least two sprays) was
applied when leaf incidence of mildew was still low (B/10% leaf infection). The early
problems related to reduced efficacy at lower RHs could be overcome by spraying in the early
morning or late afternoon, and by incorporation of a mineral oil-based surfactant as a
wetting agent (Daoust & Hofstein, 1996). AQ10 was subsequently registered for use on a
number of other fruits and vegetables in conjunction with the wetting agent, Add-Q (Paulitz
& Bélanger, 2001; Bélanger & Labbé, 2002). Currently, it is recommended as a part of an
integrated programme which includes sulphur and sterol biosynthesis inhibitor fungicides. It
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is clear that there is still a need for more development work to produce an Ampelomyces
product with reliability approaching that of conventional fungicides.
Mass production methods of some isolates were also developed in the former Soviet Union
(Puzanova, 1984; Davydova et al ., 1985) where they were subsequently commercialized
(Anonymous, 1989), one with the name Ampelomycin (Yakushkina et al ., 1994), but there is
little information available about their use, efficacy and further development.

Compatibility with Fungicides

Ampelomyces has been shown to be compatible with a large number of fungicides used in the
control of powdery mildews, such as triforine, quinomethionate (Sundheim, 1982; Sundheim
& Amundsen, 1982), triadimefon (Philipp et al. , 1982; Philipp & Kirchhoff, 1983; Shishkoff
& McGrath, 2002), myclobutanil (Shishkoff & McGrath, 2002) and other fungicides
(Sztejnberg et al ., 1989; Elad et al ., 1996). In addition, Ampelomyces also tolerates many
acaricides and insecticides (Philipp et al ., 1984), which makes it possible for their use in
integrated pest management programmes.
During commercialisation, the fungal biocontrol product AQ10 was screened against a
range of fungicides, insecticides and common surfactants used in grape-vine production. It
was not affected by certain copper-based fungicides used against downy mildew, which
allows it to be used in a single tank-mixed treatment for both diseases (Hofstein et al ., 1996).
The fact that it is not affected by sterol biosythesis inhibitors offers an opportunity to use the
fungal product as part of an integrated pest management programme using a reduced spray
regime of conventional fungicides (Hofstein & Fridlender, 1994). A benomyl-tolerant strain
of AQ10 has been induced, with no detrimental effect on biocontrol activity (Feldman et al .,
1993), which widens the applications where this anti-powdery mildew product might be used.
Interestingly, AQ10 has also been shown not to interfere with the activity of another fungal
biocontrol product, Trichodex, when co-inoculated onto cucumber for dual control of
powdery mildew and grey mould (Elad et al ., 1998).

Comparison of the Biocontrol Ability of Ampelomyces with that of other Antagonists

Cucumber powdery mildew is one of the major problems in greenhouse production that
would require a non-chemical control method. Consequently, most of the work with fungal
antagonists, including the comparison of their biocontrol efficacy, has been done against this
powdery mildew disease. The screening of 17 fungal antagonists, applied as spore
suspensions, showed that A. quisqualis, Aphanocladium album (Preuss) Gams and Tilletiopsis
albescens Gotzhale were the most promising candidates for biocontrol (Hijwegen, 1988). In a
semi-commercial-scale glasshouse trial, the biocontrol efficacy of Pseudozyma flocculosa was
shown to be superior to that of the alternatives Verticillium lecanii (Zimm) Viegas and
A. quisqualis (Dik et al ., 1998). In another experiment, isolates of Tilletiopsis spp., applied
 646 L. KISS ET AL.

24 h before inoculating cucumber plants with powdery mildew, suppressed the pathogen by
90
/97% compared to the controls, while an Ampelomyces isolate, also applied as a
prophylactic treatment, was ineffective (Hartmann et al ., 1984).
Rose powdery mildew is another major problem in greenhouse systems. Verhaar et al.
(1999) compared the efficacy of five fungal biocontrol agents (A. quisqualis, Aph. album ,
Pseudozyma rugulosa , T. minor Nyland and V. lecanii ) against this pathogen at different
RHs, and found that only one isolate of V. lecanii was able to control the disease efficiently at
a RHB/100%. According to Paulitz and Bélanger (2001), the best antagonist that suits the
practical requirements of the cucumber and rose production in greenhouses is P. flocculosa .

Efficacy of Ampelomyces as a Biocontrol Agent

Reported data on the efficacy of Ampelomyces mycoparasites are contradictory. In some
experiments, good control of powdery mildew infections of various crops was achieved using
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conidial suspensions of different isolates (Table 1) or with AQ10 (Hofstein & Fridlender,
1994; Pasini et al ., 1997; Romero et al ., 2003). Other trials showed that the biocontrol was
ineffective, with or without formulation, although parasitism of powdery mildew colonies on
the treated crops did occur (Diop-Bruckler & Molot, 1987; Yakushkina et al ., 1994; Dik et
al ., 1998; McGrath & Shishkoff, 1999; Verhaar et al ., 1999; Shishkoff & McGrath, 2002).
When compared with other antagonists in biocontrol trials, Ampelomyces isolates were less
efficient than P. flocculosa , P. rugulosa , V. lecanii , Aph. album and T. minor (Dik et al ., 1998;
Verhaar et al ., 1999). The failure of biocontrol by Ampelomyces in these experiments might
have been due to the need for high humidity, but alternatively might have reflected the slow
development of these mycoparasites in powdery mildew colonies.

CONCLUSIONS
Historically, Ampelomyces was one of the first mycoparasites to be studied in detail
(De Bary, 1870), and was also the first fungus used as a biocontrol agent of plant parasitic
fungi (Yarwood, 1932). The effect of Ampelomyces in the control of powdery mildew
infections is slow but it is still important because it suppresses the sporulation rate of its
fungal hosts, and the infected plants regain vigour after Ampelomyces has killed the
pathogens. This mycoparasite has now become one of the most advanced in terms of
commercial development of a fungal biocontrol agent for plant pathology, although there are
still problems with the efficacy of the Ampelomyces product, AQ10 Biofungicide, especially
at lower RHs. Most of the alternative fungal antagonists of powdery mildews, such as
Pseudozyma spp., Verticillium lecanii , and Tilletiopsis spp., act more quickly through the
production of antifungal compounds. However, the use of these antagonists might be
hindered by the potential side-effects of their still little known metabolites (Kiss, 2003), and
Ampelomyces might still be an appropriate candidate for future commercial development. In
addition, the relationship between host plants, powdery mildews and Ampelomyces could be
further studied from an ecological point of view to understand the role of fungal antagonists
in the natural population dynamics of plant parasites.

ACKNOWLEDGEMENTS
We thank the Royal Society for a UK-CEE joint project grant to PJ and LK which initiated a
collaboration between our institutions. This work was partially supported by an EU-INCO
project (SMADIA ICA4-2000-10011), a János Bolyai Research Fellowship awarded to LK
and a grant (OTKA F32931) of the Hungarian Scientific Research Fund.
 BIOLOGY AND BIOCONTROL POTENTIAL OF AMPELOMYCES 647

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