620
Mycol. Res. 100 ( 5 ) : 6 2 M 2 4 (1996) Printed in Great Britain
Genetic studies of the phytopathogenic fungus Botryotinia
fuckeliana (Botrytis cinerea) by analysis of ordered tetrads
F. FARETRA A N D S. POLLASTRO
Dipartimento di Protezione delle Piante dalle Malattie, University of Bari, Via G. Amendola 165/a, 70126 Bari, Italy
Botryotinia fuckeliana (Botrytis cinerea) is a heterothallic ascomycete which produces linear 8-spored asci (tetrads)in apothecia.
Ascospores were collected in their linear sequence from individual asci, and analysed to determine the segregation patterns of alleles
at gene loci controlling mating type (MATI), resistance to dicarboximide fungicides (Dafl) and resistance to benzimidazole
fungicides (Mbcr). The alleles were present in their expected linear sequence in 58% of the asci; most irregular sequences (32% of
the asci) were caused by single exchanges between adjacent spores. The MAT1 locus was ca 12 map units from its centromere, and
the Mbcl locus showed loose linkage with its centromere. The Mbcr and Dafl loci were ca 47 map units apart on the same
chromosome and not linked with the MAT1 locus. Pairs of homothallic ascospores were found in seven out of 105 asci.
Homothallism was not caused by inclusion of more than one nucleus in ascospores, and it occurred only in ascospores predicted to
carry the MATI-2 allele after meiosis.
Botyotinia fuckeliana (de Bary) Whetzel, teleomorph of Botytis
cinerea Pers., is the pathogen which causes 'grey mould'
disease on numerous economically important plant species.
The fungus became amenable to genetic analysis when a
reliable method for obtaining meiotic progeny under laboratory conditions was developed (Faretra & Antonacci, 1987;
Faretra & Pollastro, 1988; Faretra, Antonacci & Pollastro,
1988a, b). Apothecia containing linear asci are obtained by
cross-fertilization of sexually compatible isolates carrying
different alleles of the mating type gene, MATI. Each ascus is
derived from a single, heterozygous, diploid nucleus, and
contains four pairs of ascospores; the nuclei in each pair of
ascospores should be genetically identical, since they are the
result of mitotic division of one of the four products of meiosis
(Lorenz & Eichhom, 1983; Faretra & Antonacci, 1987; Faretra,
Contesini & Pollastro, unpublished).
Apothecia can be transferred to vials of sterile distilled
water, and ruptured with tweezers to release thousands of
ascospores for the analysis of random meiotic spores (Faretra
& Grindle, 1992). This method has been used to determine the
genetic basis of fungicide resistance (Faretra & Pollastro, 1991,
1993a, b; Faretra, Pollastro & Sansiviero, 1992; Pollastro &
Faretra, 1992; Pollastro ef ai., 1995), morphological differences
(Faretra & Mayer, 1992; Faretra & Pollastro, 1992), and
auxotrophy ( ~ a r e t r a& Pollastro, unpublished). However, it
cannot provide some kinds of genetic data for which tetrad
analysis is essential; for example, the data needed to locate a
gene in relation to its centromere (Burnett, 1975; Fincham ef
al., 1979).
This report deals with investigations of mating type and
resistance to benzimidazole and dicarboximide fungicides by
analysis of ascospores from individual asci of B. fuckeliana.
MATERIALS A N D M E T H O D S
Botryotinia fuckeliana isolates
The isolates used in this study (Table 1) carried known alleles
of the genes conferring differences in mating type (MATI),
response to benzimidazole fungicides (Mbcl) and response to
dicarboximide fungicides (Dafl), as determined by Faretra &
Pollastro (1991, 1993a). Alleles of these three genes were
used to detect segregation patterns in tetrads.
Sexual crosses and derivation of ascospore progeny
The following strains (sclerotial $? parent x spermatizing 6
parent) were crossed to obtain apothecia as described by
Faretra ef al. (1988a): SAS405 x SAS56 (cross CRI),
SAS405 x 2A/12 (CR2), SAS405 x C/4 (CR3), SAS405 x
2E/5 (CR4), SAS405 x G / I (CR5), SAS56 x SAS405 (CR6),
SAS56 x 2B/1 (CR7), SASS6 x 2D/1 (CRS), SAS56 x F/1
(CR9), SAS56 x 2H/1 (CRIO). Intact asci were transferred
individually to dishes of water agar (20 g Oxoid technical agar
1-1 d'istllled
'
water), and the ascospores were dissected out in
their linear sequence with a Leitz micromanipulator to obtain
ordered 8-spored tetrads. Germinated ascospores were grown
on slants of malt extract agar (MEA, 20 g Oxoid malt extract
and 20 g Oxoid technical agar I-' distilled water) to obtain
colonies.
Genotypes of ascospore progeny
Resistance or sensitivity of the ascospore progeny to
fungicides was determined by transferring hyphal inocula to
dishes of MEA containing benomyl or diethofencarb to detect
F. Faretra and S. Poflastro
62 1
Table 1. Origins and genotypes of strains of Botryotinia fuckeliana used for
tetrad analysis
Genotypes
Origin (9x parents
in sexual crosses)*
Straint
-
SAS56
2A/I2
G/1
c/4
2E/5
SAS405
2H/I
2D/I
28/1
F/I
-
MAT1
Mbcl
Daf1
I
I
S
S
S
LR
HR"
HR*
S
LR
HR*
HR'"
S
S
HR
S
S
LR
HR
S
LR
LR
-
WS55 x unknown
SAS405 x SAS56
V29 x SAS56
SAS56 x 813
SAS405 x VB74
WS158 x unknown
V29 x SAS56
SAS56 x 813
SAS56 x SAS405
SAS405 x VB74
1
1
I
2
2
2
2
2
t Each strain was a single ascospore isolate from the cross indicated.
All parental strains excepting SAS were field-isolates.
5 S, sensitivity; LR, low resistance; HR, high resistance.
+
" Alleles also conferring hypersensitivity to diethofencarb.
** Alleles also conferring normal sensitivity to diethofencarb.
alleles of the Mbc1 gene, and MEA containing vinclozolin to
detect alleles of the Dafl gene, as described by Faretra &
Pollastro (1991). The mating types of the progeny were
determined by crossing each one to both the MATI-1
reference strain, SAS56, and the M A T I - 2 reference strain,
SAS405 (Faretra et al., 1988b). Genetic symbols and
terminology for B. fuckeliana isolates have been described by
Faretra & Grindle (1992).
Tetrad analysis
Sequences of genotypes in individual asci were deduced from
segregations of genes conferring differences in fungicide
resistance. The sequences so obtained could often be
corroborated by segregations of morphological traits of
colonies; for instance, conidiation, number and size of sclerotia.
Pairs of ascospore progeny with identical phenotypes were
easily recognized in most tetrads.
Ordered asci were analysed to determine whether the
different alleles at a gene locus segregated from each other at
the first meiotic division (FDS; asci with a 4:4 arrangement of
alleles in the spores) or the second meiotic division (SDS; asci
with a 2:2 :2: 2 arrangement of the alleles). Generally, in
absence of particular modality of meiotic recombination, a
proportion of SDS :FDS lower than 2 :1indicates that the gene
locus is linked to its centromere, and 50% of the frequency of
SDS asci is an estimate of their map distance (Fincham et al.,
1979).
Ordered and unordered asci were analysed for recombination between pairs of gene loci. Asci were classified as
parental ditypes (PD) containing only parental genotypes;
non-parental ditypes (NPD) containing only recombinant
genotypes, and tetratypes (T) containing both parental and
recombinant genotypes. The proportion of PD:NPD asci
indicates whether the gene loci are on the same or different
linkage groups (Fincham et al., 1979). Genes assigned to the
same linkage group were mapped using the formula
50[(T 6NPD)/(PD NPD T)], including the correction for
double crossovers (Fincharn et al., 1979).
+
+
+
Whether there was independent segregation of genecentromere and gene-gene loci was assessed by means of the
X2 test incorporating the Yates correction term (Sachs, 1982).
RESULTS A N D DISCUSSION
Alleles of the Mbc1 and Dafl genes segregated in a normal
1: 1ratio in all asci from all crosses (167 asci were analysed for
the Mbcl gene and 158 asci for the Daf1 gene). The
M A T I - 1 and M A T I - 2 alleles of the mating type gene also
segregated normally in a I :1ratio in most (98 out of 105) asci
examined. The seven exceptional asci contained homothallic
ascospores (see below).
The sequences of genetic markers were determined in 190
asci (Table 2). Regular, normal, linear sequences (type I )
occurred in 110 asci (58%). Most of the irregular sequences
(types 2 and 3), observed in 61 asci (32%), were due to single
exchanges between two adjacent spores. Only 19 asci (10%)
had irregular sequences which implied more complex spore
displacements (types 4-7). The high frequency of irregular
spore sequences in mature asci can be explained by cytological
studies of meiosis in developing asci of B. fuckeliana (Faretra,
Contesini & Pollastro, unpublished). Those showed that the
nuclear spindles of the first post-meiotic mitosis often overlap
to their oblique orientation in the ascus, and they may not be
parallel. The overlapping spindles which could be responsible
for irregular sequences in ascus types 2-7 are illustrated in
Table 2.
The frequencies of SDS tetrads in regard to the Mbcl, Dafl
and M A T 1 gene loci were 58, 72 and 25 % respectively (Table
3). Results of x2tests (Table 3) show that only the M A T 1 gene
is linked to its centromere and can be mapped (12.5 map
units); the Mbc1 gene may also be loosely linked to its
centromere.
The number of PD, NPD and T tetrads relating to
segregations of the three possible gene pairs (Table 4) show
that the Mbcl and Dafl genes are probably linked, since the
PD:NPD ratio differs significantly from 1:l. The calculated
distance between the two genes is ca 47 map units. PD :NPD
~ the gene
ratios were not significantly different from 1 : for
pair M A T I - M b c l and MATT-Dafl (Table 4), and so these
genes are probably unlinked. These conclusions are in
agreement with those based on analysis of random ascospore
progeny
(Faretra & Pollastro, 1991, 1992).
.
Seven asci from four different crosses contained ascospores
which were fertile with both M A T I - 1 and M A T I - 2 reference
strains. Strains of this phenotype have been referred to as
homothallic MATI-112 strains (Faretra & Grindle, 1992). In
five of these asci, there were two pairs of MATI-1 ascospores
and two pairs of MATI-112 ascospores; in the other two asci,
there were two pairs of MATI-I, one pair of MATI-2 and
one pair of MATI-112 ascospores. Thus, pairs of homothallic
MATI-112 ascospores replaced pairs of MATI-2 ascospores.
Since alleles of the Mbc1 gene and/or the Dafl gene
segregated in these asci as ~redictedon the basis of parental
genotypes, it seems very unlikely that the abnormal mating
type ascospores were due to contamination or experimental
errors during fertilization.
Previous studies of field isolates and monoascospore isolates
Genetics of Bofryofinia fuckeliana
622
Table 2. Numbers of ordered asci of Botryotinia fuckeliana in which the ascospores display regular and irregular sequences of marker genes
Ascus
type?
Arrangement of ascospores
in ascit
crosses5
CRI
CR2
CR3
CR4
CR5
CR6
CR7
CR8
CR9
CRlO
Total
asci
1
0-0
Q-Q 0-0@-@
42
10
8
8
4
8
6
5
13
6
110
4
0Q-Q @ 0-0@-@
5
I
I
o
o
o
o
o
o
o
7
82
18
16
9
8
13
II
7
18
8
190
Total asci
t Type I shows the regular linear sequence of marker genes in the nuclei of eight spores. Types 2-7 are irregular sequences caused by overlapping
nuclear spindles.
Pairs of spores carrying identical genetical markers are coded with the same number. Ascus polarity is ignored. The markers were alleles of genes
encoding differences in fungicide resistance and morphology. There should be four pairs of spores with identical genotypes since they are produced by
post-meiotic mitosis of the four products of meiosis. Lines indicate overlapping nuclear spindles (during post-meiotic mitosis) which can account for each
type of sequence irregularity.
6 Asci were obtained from apothecia of the ten CR crosses described in the text.
Table 3. Numbers of ordered asci (tetrads) from crosses of Botyotinia
fuckeliana strains showing particular types of segregation of alleles at
three gene locit
Gene locus*
Mbcl
Daf I
Cross
FDS
SDS
FDS
CRI
CRZ
CR3
CR4
CR5
CR6
CR7
CR8
CR9
CRIO
Total asci
Frequency (%)
value5
Probability
level (P)
33
7
7
2
41
18
6
x2
8
5
7
4
4
6
8
3
6
3
5
5
13
na
na
70
97
42
58
5.61
< 0.02
2
na
2
6
2
na
6
2
44
28
MAT1
SDS
55
9
10
na
6
8
8
na
I2
6
114
72
1.65
> 0.19
FDS
32
7
3
5
3
9
4
4
9
3
79
75
SDS
8
2
5
0
3
I
I
3
I
2
26
25
83.08
< 0.001
t Alleles of the gene segregated from each other at the first meiotic
division (FDS) or second meiotic division (SDS). SDS tetrads are due to
recombination between the gene locus and its centromere.
Responsible for difference in resistance to benzirnidazole fungicides
(Mbcl), resistance to dicarboximide fungicides (Dafl) and mating type
(MATI). na, not applicable.
5 For independent gene-centromere segregation the null hypothesis
tested is SDS:FDS = 2: 1.
*
of B. fuckeliana (Faretra ef al., 1988b) have identified
functionally homothallic strains. Inclusion of two nuclei of
opposite mating type in one ascospore is the rule in the fourspored asci of secondarily homothallic species such as
Neurospora fefrasperma (Dodge, 1927) and Podospom anserina
(Franke, 1957). A similar mechanism has been shown to
operate in functionally homothallic field isolates of B. fuckeliana
since they were shown to be heterokaryons containing both
A4ATl-1 and M A T I - 2 alleles in separate nuclei (Faretra ef al.,
19886). That is, they are secondarily (pseudo) homothallic.
However, homothallism of monoascospore isolates cannot be
due to heterokaryosis and has not been explained. Cytological
observations have shown that ascospores incorporate a single
post-meiotic nucleus (Lorenz & Eichhom, 1983; Faretra &
Antonacci, 1987; Faretra, Contesini & Pollastro, unpublished);
consequently, they are presumably not heterokaryotic. Also,
homothallic ascospores have been detected in asci with eight
viable ascospores and with the expected segregation of
phenotypes. Irregular inclusion of more than one nucleus of
opposite mating type in individual ascospores is then very
unlikely since it would be expected to result in aborted
(anucleate) spores in eight-spored asci.
Molecular studies of mating type genes in Ascomycetes
have shown that unique DNA sequences are associated with
specific alleles (reviewed by Glass & Lorimer, 1991; Glass &
Nelson, 1994). The sequence differences between alleles
(idiomorphs, according to Metzenberg & Glass, 1990) are so
extensive that changes from one allele to another do not
occur; furthermore, no new alleles have been produced
experimentally by mutation or intragenic recombination
F. Faretra and S. Pollastro
623
Table 4. Numbers of ordered asci (tetrads) from crosses of Botyotinia fuckeliana strains showing particular types of segregation of gene pairst
Gene-gene segregation
Cross
PD
NPD
CRI
CR2
CR3
CR4
CR5
CR6
CR7
CR8
CR9
CRlO
Total asci
Frequency (%)
X' value for independent
segregation*
Probability level (P)
28
4
4
na
7
0
0
na
0
0
0
na
0
na
7
4
25.41
1
3
1
na
3
na
44
26
T
PD
NPD
T
PD
NPD
T
50
14
12
na
7
I1
9
na
15
na
118
70
7
3
2
0
0
3
3
2
I
na
21
21
13
20
5
6
5
6
6
2
4
8
na
62
62
5
4
0
na
3
I
3
na
2
32
1
0
0
0
I
0
1
1
na
17
17
0.24
1
3
2
na
I
0
16
17
1
0
na
1
4
5
na
3
6
3
na
8
0
5
11
66
71
12
0.59
t PD, parental ditypes (only parental genotypes present); NPD, non-parental ditypes (only non-parental genotypes present); T, tetratypes (both parental
and non-parental genotypes present). na, not applicable.
$ The null hypothesis tested is PD:NPD = 1:I.
(Newmeyer et al., 1973; Griffiths, 1982). Therefore, it is
unlikely that homothallism of B. fuckeliana ascospores is due to
simple mutation from the MATI-2 to the A4ATI-1 allele.
Random ascospore progeny from crosses of two homo~ ~S A S ~with
~ , the reference SAS405
thallic isolates, S A S and
(MATI-2), were back-crossed to both strains SASS6 (MATII) and SAS405 (MATI-2) in order to determine their mating
types. Most of the 128 ascospores tested were heterothallic
(59 were MATI-1 and 63 were MATI-2). Only six ascospores
were homothallic MATI-112. Thus, homothallism of isolates
SAS27 and SAS39 was not caused by a defective MAT1 gene
nor by a stable 'homothallic' locus and they acted as MATI-1
partner in the cross with SAS405.
Mating type instability has been documented in several
species of fungi- In the case of the heterothallic yeasts,
Saccharomyces cereuisiae and Schizosaccharomyces M om be, the
molecular mechanism of mating type switching is known
(Beach & Klar, 1984; Herskowitz, 1988), Perkins (1987)
suggested that a similar mechanism might be responsible for
unidirectional reversal of mating type observed in some
filamentous Ascomycetes, including Chromocrea spinulosa
(Mathieson, 1952), Fusarium subglufinans (Leslie ef al., 1986),
Glomerella cingulafa (Wheeler, 1950) and Sclerotinia trifoliorum
(Uhm & Fujii, 1983). Turgeon, Christiansen & Yoder (1993),
however, observed that the phenomenon is uncommon and
has not been found in any of the well-studied species of
filamentous fungi. In these organisms, a unique copy of the
mating-type genes is present in the genome so that
homothallism through a switching mechanism as in yeasts is
prevented.
Mating type instability is now well documented in
B. fuckeliana, since homothallic isolates of the fungus have
been detected in three separate studies, representing ca 5 % of
the total random ascospores analysed (Faretra ef al., 19886;
Faretra & Pollastro, 1991; this investigation). This study has
shown that the mating type change in B. fuckeliana always
involves the A4ATI-2 allele and is apparently
unidirectional. It
*.
should be possible to explore further the genetic and molecular
bases of mating type instability by cloning and sequencing the
MATI-I and A4ATI-2 idiomorphs.
This investigation was supported by a grant from Consiglio
Nazionale delle Ricerche to the project 'Genetics of Bofyotinia
fuckeliana (Bofyfis cinerea)' which is part of the co-ordinate
research programme 'Fungal genetics'. We wish to thank Dr
Morris Grindle (The University of Sheffield, U.K.) for critical
reading of the manuscript.
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