ESSENTIAL! To study thalli, trichospores, etc., Harpel-
lales can be grown in small (6015 mm) petri dishes.
After the one-tenth BHIv agar solidifies, pour a very thin
layer of sterile, distilled water over the medium sufficient
to cover the surface. Small petri dishes are recommended
because they are easier to manipulate than larger ones.
All cultures should be stored in the refrigerator
soon after good growth has occurred, preferably in
screw-capped test tubes. Most isolates must be trans-
ferred every 4 months and slow-growing cultures every
2–3 months. Storage in liquid nitrogen is the best
method for long-term preservation using conventional
techniques for freezing and thawing. USE YOUNG,
ACTIVELY GROWING THALLI (NOT SPORES). Do
NOT lyophilize cultures of Harpellales!
With few exceptions, trichospores of Har-
pellales do not germinate in vitro. This necessi-
tates the transfer of portions of thalli that are
broken into smaller pieces in the new culture
media. This is best done with a standard bacte-
riological transfer loop.
In slants, the loop should be rapidly and vigorously
agitated sideways in the water laver at the bottom of
the tube until the thalli are broken up sufficiently. In
petri dishes, the loop can be agitated sideways in the
water layer or vibrated by dragging the wire of the loop
back and forth over the upper edge of the plastic petri
dish bottom. For more precise handling of fungal mate-
rial in petri dishes, a small loop (4–5 mm in diameter) is
preferable to the more standard size.
Thalli developing in petri dishes can be left undis-
turbed when new growth is being produced. In test
tubes, however, the slants should be tilted daily to
allow the water to flow over the surface of the culture
medium. This should be done for 3–7 days and stopped
when good growth of new thalli is observed on the slant
both above and below the surface of the water. Some
taxa grow best at 24C, but others have their optimum
growth temperature near 18C.
Cultures of many zygomycetes are available
from the American Type Culture Collection
(ATCC: Manassas, Virginia,USA), Centraalbur-
eau voor Schimmelcultures (CBS: Utrecht, the
Netherlands), International Mycological Institute
(IMI:Egham,UK),andtheUSDA-ARSCulture
Collection (NRRL: National Center for
Agricultural Utilization Research, Peoria, Illinois,
USA). Entomopathogenic Entomophthorales are
available from the USDA-ARS Collection of
Entomopathogenic Fungal Cultures (ARSEF:
Ithaca,NewYork,USA;Humber2012b).
VIII. Conclusions
The conidiogenesis of the Entomophthoromy-
cota and some members of the Zoopagomyco-
tina should be regarded as fundamentally
distinct from the sporangiogenesis in Mucor-
ales and related orders,from the production of
cylindrical sporangia in Kickxellales, Dimar-
garitales, and several taxa of Zoopagales, and
from the highly modified monosporic sporan-
giola of Asellariales and Harpellales. Similarly,
the relatively undifferentiated state of the con-
idiogenesis of Entomophthoromycota and Zoo-
pagomycotina is markedly different from the
obviously sporangiate zygomycetes whose
asexual reproductive systems are usually well
differentiated (often elaborately so) from the
vegetative mycelium. Members of Ento-
mophthoromycota are further distinguished
from the remaining zygomycotan fungi by
strictly homothallic zygosporogenesis and a
marked (secondary?) tendency for azygosporo-
genesis as opposed to the well-differentiated
and morphologically complex heterothallic
zygosporogenesis in the sporangiate zygomy-
cetes and most members of Zoopagales.
The phylogeny of the zygomycotan fungi
does not conform to the simpler unitary
schemes of past decades based on morphology,
development, and biology. The initial phyloge-
netic studies suggested that members of Ento-
mophthorales were paraphyletic, with
Basidiobolusbeing notably distinct from the
remainder of the phylum (Jensen et al. 1998 ;
Nagahama et al. 1995 ). The recent papers by
Gryganskyi et al. ( 2012 , 2013 ) clearly demon-
strate thatBasidiobolusis a member of the
Entomophthoromycota.
Initially, only a few zygomycetes were ever
included in a phylogenetic analysis. These
treatments have usually included small subunit
rDNA (Sugiyama 1998 ), but there has never
been enough sequence information available
for these analyses to provide an accurate repre-
sentation of the phylogeny of the zygomycetes.
Recent studies, however, have included many
more genes in the data sets used for phyloge-
netic analysis (Gryganskyi et al. 2012 , 2013 ;
James et al. 2006 ; White et al.2006a).Zygomycetous Fungi: Phylum Entomophthoromycota and Subphyla Kickxellomycotina,... 239