Introduction
Sclerotinia sclerotiorum, is one of the most important pathogens
of soybean worldwide. This pathogen causes the white mold disease in a broad
host range. Disease management is complicated by the long-term survival of sclerotia in the soil and the absence of resistance in
elite, commercial cultivars (Willbur et al.
2019).
It is known that S. sclerotiorum isolates originating from different
geographical areas and hosts may vary in morphological, physiological and
genetic characteristics (Kull et al. 2004; Durman et al. 2005; Li et
al. 2008; Garg et al. 2009). The expansion of the genetic
diversity of most living organisms occurs by means of sexual reproduction
(which is meiosporic in the case of fungi), but to
date this type of reproduction has not been described for certain fungal species
(Carlile et al. 2001). In contact areas of colonies
of a same isolate or between different isolates of the same fungal species, the
occurrence of sexual compatibility (sexual reproduction) and vegetative
compatibility or incompatibility can be observed (Glass et al. 2000). Hyphal anastomosis allows the exchange of cell contents
between different individuals in compatibility reactions, the onset of sexual
reproduction, heterokaryon formation, occurrence of a
parasexual cycle and nutrient distribution in the
mycelium/colony (Glass et al. 2000; Hickey et al. 2002).
Based on the genetic
description, fungi are called antagonistic when clearly distinguishable sexual
and vegetative heterokaryons occur at the interface
between hyphae of different isolates (Alexopoulos et
al. 1996). Sexual reproduction can occur among both heterothallic and
homothallic isolates (Robertson et al. 1998). The
test of mycelial compatibility groups has been used
to evaluate the genetic diversity in several plant-pathogenic fungi (Katan et al. 1991; Leslie
1993), including S. sclerotiorum
(Kull
et al. 2004; Li et al. 2008).
Population studies of S.
sclerotiorum demonstrated the genetic diversity
and provided evidence for clonal and/or sexual reproduction. Generally, clonal
reproduction predominates in temperate regions (Hambleton et al. 2002), while sexual recombination occurs in warmer climate regions (Malvárez et al. 2007). Kohn et al. (1990) studied mycelial
compatibility among 35 S. sclerotiorum isolates
from different regions of Canada, and clustered the isolates in 28 groups of mycelial compatibility, 23 isolates were incompatible with
all and 12 isolates formed compatibility groups with 2–3 isolates, indicating
high genetic heterogeneity within the species. The reported degree of mycelial incompatibility suggests that genetic exchange
occurs eventually in fungal populations. However, this exchange is more likely
to occur through the ascus in sexual reproduction,
since the high mycelial incompatibility between S.
sclerotiorum isolates probably decreases the
possibility of somatic heterokaryosis through hyphal fusion.
In Brazil, the formation of five
mycelial compatibility groups and three clusters was
detected by RAPD, suggesting moderate genetic diversity and sexual recombination
among isolates from the tropics (Júnior et al. 2011).
Meinhardt et al. (2002) reported two
compatibility groups among 23 isolates. Differences between
groups of mycelial compatibility as the
aggressiveness of soybean plants were observed by Kull et al. (2004).
Despite the decades-old presence
of S. sclerotiorum in Brazil,
information about the population biology of this pathogen as well as on the
aggressiveness of isolates of this fungus on soybean plants are still scarce,
especially with regard to the Midwest region. The objectives of the present
study were to investigate the genetic diversity in S. sclerotiorum populations on soybean in Central Brazil
by the analysis of mycelial compatibility, and to assesse the aggressiveness of isolates in mycelial compatibility groups of soybean plants.
Materials and Methods
Population of S. sclerotiorum
Nine populations of S.
sclerotiorum isolates were derived from sclerotia collected from eight soybean-producing regions in
the Central Brazil. Sclerotia were collected from
each location at approximately 50 points, then selecting 25 isolates per
sampled field. The distance between the sampling points in each field was at
least 10 m; only in the municipality of Silvânia, Goiás, two samples were collected from two fields on the
same farm.
To obtain the isolates in the mycelial form, the sclerotia were
sterilized in 96% alcohol and 2% sodium hypochlorite for 60 s in each solution
and then placed on Petri dishes containing agar-water culture medium and
incubated in a chamber, in constant darkness at 20 ± 1°C. After initiation of sclerotia germination, 6-mm diameter discs containing only
tips of the fungal hyphae were removed from the edges of the colony and placed
on Petri dishes containing PDA (potato dextrose agar) culture medium and incubated in a
chamber under the above conditions.
Intrapopulation analysis of mycelial compatibility of S. sclerotiorum isolates
The experiments were conducted
in a completely randomized design with two replications and 25 isolates per
population, with one replication cultured in MPM (modified Patterson's medium)
and the other in PDA (potato dextrose agar) culture medium. Two culture media
were used, as in a previous study it was found that, in some cases, the MPM
medium delays the growth of fungi, making evaluation difficult and inducing
errors. The inhibition of mycelial growth was
observed by Schafer and Kohn (2006) in the presence of MPM medium. Both culture
media were used for greater insight and reliability in the assessments of mycelial reactions.
The MPM culture medium was
prepared as proposed by Schafer and Kohn (2006), containing:0.68 g L-1
KH2PO4; 0.50 g L-1 MgSO4.7H2O;
0.15 g L-1 KCl, 1.0 g L-1 NH4NO3;
18.4 g L-1 D-glucose; 0.50 g L-1 yeast extract; 15.0 g L-1
agar; 200 μL solution of trace elements
containing 95 mL distilled water; 5.0 g monohydrate citric acid; 5.0 g ZnSO4.7H2O;
1.0 g Fe(NH4)2(SO4)2.6H2O;
0.25 g CuSO4.5H2O; 0.05 g MnSO4.1H2O;
0.05 g H3BO4; 0.05 g Na2MoO4.2H2O;
and 1 mL CHCl3 as a preservative solution stored at 20 ± 2°C. The
PDA culture medium was prepared as described by Zauza
et al. (2007), containing 20 g L-1 D-glucose, 17 g L-1 agar
and 200 g potato. Before pouring the culture media into Petri dishes, 1.000 μL of strawberry red dye (Mix Coralim®) were
added to facilitate the visualization of incompatibility reactions.
The isolates were cultured in
PDA for 5 d in the dark at 25 ± 1°C. After this period, 6-mm diameter mycelial discs were removed from the edge of the S.
sclerotiorum colony and three discs were placed
equidistantly in each Petri dish (diameter 9 cm), containing culture medium.
After this, the Petri dishes were incubated in a BOD chamber in the dark at 25
±1°C. The mycelial interaction reactions were
evaluated 7 d after incubation, considering a reaction incompatible when a red
line was detected and formation of aerial mycelium at the interface line.
Reactions were considered compatible when the colonies grew freely in the
culture medium and were fused.
Interpopulation analysis of mycelial compatibility of S. sclerotiorum isolates
Two isolates from each group of mycelial compatibility of each location were paired with
each other, amounted to 31 isolates for interpopulation
analysis. Some mycelial compatibility groups were
represented by a single isolate since they were comprised of a solitary
isolate. This experiment was conducted similarly to that described above. The
results showed that the compatibility groups were distinct or similar to the
groups already determined within each population/location.
Aggressiveness of S. sclerotiorum isolates in soybean
In the study of aggressiveness
of S. sclerotiorum isolates on soybean,
a single isolate from each compatibility group detected for each location was
randomly chosen, totaling 21 isolates. These isolates (SSMO01, SSMO23, SSUB01,
SSUB18, SSAF11, SSSI37, SSSI21, SSSI16, SSSI10, SSPA11, SSPA03, SSPA23, SSCS24,
SSCS05, SSAN02, SSAN11, SSAN20, SSSM12, SSSM25, SSSM03 and SSSM10) were
evaluated for aggressiveness on the cultivars M-SOY 7908 RR and BRSGO 7760 RR, since
these two cultivars have been reported as highest and lowest disease incidence,
respectively, among commercial varieties under field conditions in central
region of Brazil. The cultivars were sown in 5 L pots filled with Oxisol soil under greenhouse conditions. The experiment was
repeated once to verify the consistency of results.
The experiment was conducted in
a completely randomized, factorial design with 21 (isolates) × 2 (cultivars),
with 3 replications. Each replication consisted of five plants, i.e., a total
of 15 plants per treatment. When the plants reached stage V3/V4, the third
trifoliate leaf was cut with scissors, leaving about 3 cm of the stem for the
insertion of the inoculum. For the stem inoculation, the entire length of 1000 µL
plastic tips was filled with mycelial discs taken
from the edge of inverted S. sclerotiorum
colonies such that the upside down mycelium came to direct contact with the
plant at the inoculation site.
The 21 isolates used in
inoculations were grown in PDA culture medium for 3 d at 25 ± 1°C and a
photoperiod of 12 h. Inoculated plants were maintained in a greenhouse for 7 d,
at a mean temperature of 22°C and relative humidity of 90%. Evaluations
consisted of measuring the lesion length on the stems, 3 and 7 d after
inoculation. Subsequently, the average lesion length of five plants per
replication was calculated.
Fig. 1: Paired S. sclerotiorum
isolates reacting with incompatibility (A,
B, C and D), compatibility
(E and F)
Data from both experiments were
subjected to normality and homogeneity tests of variance, using the SAS package
(Statistical Analysis System) (SAS Institute 1999). Statistical tests were
performed using Box-Cox power transformation (Box and Cox 1964). Once the
statistical assumptions were met, analysis of variance was performed with the F
test at 5% probability. The isolate means were compared by the Scott-Knott test
and for cultivars; the Tukey test was applied at 5%
probability, using the statistical program SISVAR (Ferreira 2000).
Results
Mycelial compatibility of S. sclerotiorum
isolates
The pairing of the isolates in
all possible combinations for each population produced incompatible and
compatible reactions. The incompatible reaction was characterized by the
presence of a red line, which were recorded from the top or the bottom of the
colonies (Fig. 1A and B) and formation of aerial mycelium along the contact
line of the isolates (Fig. 1C and D). The compatible response was defined as
the absence of a line in the contact zone of the isolates (Fig. 1E and F). The
results of the isolate compatibility analysis for each population are shown in
Table 1.
A single mycelial compatibility group (MGC1)
was detected in both Água Fria-GO population and Silvânia-GO “B” population, denoting the existence of one
clone in each sampled field (Table 1). In contrast, in the populations of Uberlândia-MG and Montividiu-GO,
two compatibility groups per population were found. Group MCG1 was also present
at these two locations, comprising 96% of the isolates, while the groups MCG7
in the population of Montividiu and MCG8 in the
population of Uberlândia were represented by a single
isolate (Table 1).
Table 1: Mycelial
compatibility groups of S. sclerotiorum isolates
detected in different municipalities
MCG |
|
|
|
|
Isolates |
|
|
|
|
Água Fria |
Silvânia "A" |
Silvânia “B” |
Montividiu |
Uberlândia |
Patrocícnio |
Anápolis |
Chapadão do Sul |
São Miguel do Passa Quatro |
|
|
SSAF01 |
SSSI01 |
SSSI26 |
SSMO01 |
SSUB01 |
SSPA01 |
SSAN01 |
SSCS02 |
SSSM01 |
|
SSAF02 |
SSSI02 |
SSSI27 |
SSMO02 |
SSUB02 |
SSPA02 |
SSAN03 |
SSCS03 |
SSSM02 |
|
SSAF03 |
SSSI03 |
SSSI28 |
SSMO03 |
SSUB03 |
SSPA03 |
SSAN04 |
SSCS07 |
SSSM03 |
|
SSAF04 |
SSSI04 |
SSSI29 |
SSMO04 |
SSUB04 |
SSPA04 |
SSAN05 |
SSCS09 |
SSSM04 |
|
SSAF05 |
SSSI05 |
SSSI30 |
SSMO05 |
SSUB05 |
SSPA05 |
SSAN06 |
SSCS13 |
SSSM05 |
|
SSAF06 |
SSSI06 |
SSSI31 |
SSMO06 |
SSUB06 |
SSPA06 |
SSAN07 |
SSCS14 |
SSSM06 |
|
SSAF07 |
SSSI07 |
SSSI32 |
SSMO07 |
SSUB07 |
SSPA07 |
SSAN08 |
SSCS17 |
SSSM07 |
|
SSAF08 |
SSSI08 |
SSSI33 |
SSMO08 |
SSUB08 |
SSPA08 |
SSAN09 |
SSCS24 |
SSSM08 |
|
SSAF09 |
SSSI09 |
SSSI34 |
SSMO09 |
SSUB09 |
SSPA09 |
SSAN10 |
SSCS25 |
SSSM09 |
|
SSAF10 |
SSSI10 |
SSSI35 |
SSMO10 |
SSUB10 |
SSPA10 |
SSAN12 |
- |
SSSM11 |
1 |
SSAF11 |
SSSI11 |
SSSI36 |
SSMO11 |
SSUB11 |
SSPA12 |
SSAN13 |
- |
SSSM12 |
|
SSAF12 |
SSSI12 |
SSSI37 |
SSMO12 |
SSUB12 |
SSPA13 |
SSAN14 |
- |
SSSM13 |
|
SSAF13 |
SSSI13 |
SSSI38 |
SSMO13 |
SSUB13 |
SSPA14 |
SSAN15 |
- |
SSSM14 |
|
SSAF14 |
SSSI14 |
SSSI39 |
SSMO14 |
SSUB14 |
SSPA15 |
SSAN16 |
- |
SSSM15 |
|
SSAF15 |
SSSI15 |
SSSI40 |
SSMO15 |
SSUB15 |
SSPA16 |
SSAN17 |
- |
SSSM16 |
|
SSAF16 |
SSSI17 |
SSSI41 |
SSMO16 |
SSUB16 |
SSPA17 |
SSAN18 |
- |
SSSM17 |
|
SSAF17 |
SSSI18 |
SSSI42 |
SSMO17 |
SSUB17 |
SSPA18 |
SSAN19 |
- |
SSSM18 |
|
SSAF18 |
SSSI19 |
SSSI43 |
SSMO18 |
SSUB19 |
SSPA19 |
SSAN20 |
- |
SSSM19 |
|
SSAF19 |
SSSI20 |
SSSI44 |
SSMO19 |
SSUB20 |
SSPA20 |
SSAN21 |
- |
SSSM20 |
|
SSAF20 |
SSSI22 |
SSSI45 |
SSMO20 |
SSUB21 |
SSPA21 |
SSAN22 |
- |
SSSM21 |
|
SSAF21 |
SSSI23 |
SSSI46 |
SSMO21 |
SSUB22 |
SSPA22 |
SSAN23 |
- |
SSSM22 |
|
SSAF22 |
SSSI24 |
SSSI47 |
SSMO22 |
SSUB23 |
SSPA24 |
SSAN24 |
- |
SSSM23 |
|
SSAF23 |
SSSI25 |
SSSI48 |
SSMO24 |
SSUB24 |
SSPA25 |
- |
- |
SSSM24 |
MCG |
Água Fria |
Silvânia “B” |
Montividiu |
Uberlândia |
Silvânia “A” |
Patrocínio |
Anápolis |
Chapadão do Sul |
S. M. Passa Quatro |
1 |
SSAF24 |
SSSI49 |
SSMO25 |
SSUB25 |
- |
- |
- |
- |
- |
SSAF25 |
SSSI50 |
- |
- |
- |
- |
- |
- |
- |
|
2 |
- |
- |
- |
- |
SSSI21 |
- |
SSAN02 |
- |
- |
- |
- |
- |
- |
- |
- |
SSAN11 |
- |
- |
|
- |
- |
- |
- |
- |
- |
SSAN25 |
- |
- |
|
3 |
- |
- |
- |
- |
SSSI16 |
- |
- |
SSCS01 |
- |
- |
- |
- |
- |
- |
- |
- |
SSCS04 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS05 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS06 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS08 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS10 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS11 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS12 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS15 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS16 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS18 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS19 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS20 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS21 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS22 |
- |
|
- |
- |
- |
- |
- |
- |
- |
SSCS23 |
- |
|
4 |
- |
- |
- |
- |
- |
SSPA11 |
- |
- |
SSSM25 |
5 |
- |
- |
- |
- |
- |
SSPA23 |
- |
- |
|
6 |
- |
- |
- |
- |
- |
- |
- |
- |
SSSM10 |
7 |
- |
- |
SSMO23 |
- |
- |
- |
- |
- |
- |
8 |
- |
- |
- |
SSUB18 |
- |
- |
- |
- |
- |
In soybean fields sampled in Patrocínio-MG and
Silvânia-GO “A”, three compatibility groups per
population were detected. In both populations of Patrocínio and Silvânia
"A", group MCG1 was represented by 23 isolates (92%), while MCG2 and
MCG3, in the population of Silvânia, and MCG4 and
MCG5 in the population of Patrocínio were constituted
of a only one isolate (4%) (Table
1). In the population of Anápolis, group MCG1
comprised 88% of the isolates, while MCG2 contained only 12% (Table 1).
In the population of Chapadão do Sul, two compatibility groups, MCG1 and MCG3, were
identified, consisting of 64 and 26% of the isolates, respectively (Table 1).
In the population of São Miguel do Passa Quatro-GO, there were three mycelial
compatibility groups: MCG1, consisting of 23 isolates (92%), and MCG4 and MCG6,
represented by a single isolate (4%).
The results of the mycelial compatibility
tests revealed low genetic diversity in S. sclerotiorum
populations within each sampled field (intrapopulation)
at the eight sampled municipalities, except for São Miguel do Passa Quatro-GO, Silvânia-GO, “A” and Patrocínio-MG,
where three compatibility groups were found, although two were represented by a
only one isolate. This indicates that the frequency of sexual recombination in
these populations, if existent, is very low.
The results of the compatibility test among the sclerotia
sampling locations are shown in Table 2. When one or two isolates representing
each compatibility group of all locations were paired in all possible
combinations, eight compatibility groups were detected (MCG1, MCG2, MCG3, MCG4,
MCG5, MCG6, MCG7, and MCG8, represented by, respectively, 19 (61.6%), 3 (9.6%),
3 (9.6%), 2 (6.4%), 1 (3.2%), 1 (3.2%), 1 (3.2%), and 1 isolates (3.2%) (Table 2).
Table 2: Isolates and mycelial compatibility groups
identified by interpopulation analysis of 31 S. sclerotiorum isolates collected on soybean fields in
the Central region of Brazil
Isolates |
Location/Population |
MCG |
SSSI37 |
Silvânia "B" |
1 |
SSSI48 |
1 |
|
SSUB01 |
Uberlândia |
1 |
SSUB18 |
8 |
|
SSUB21 |
1 |
|
SSAF03 |
Água Fria |
1 |
SSAF11 |
1 |
|
SSMO23 |
Montividiu |
7 |
SSMO01 |
1 |
|
SSMO20 |
1 |
|
SSSI21 |
Silvânia “A” |
2 |
SSSI16 |
3 |
|
SSSI10 |
1 |
|
SSSI02 |
1 |
|
SSPA11 |
Patrocínio |
4 |
SSPA23 |
5 |
|
SSPA17 |
1 |
|
SSPA03 |
1 |
|
SSAN02 |
Anápolis |
2 |
SSAN11 |
2 |
|
SSAN21 |
1 |
|
SSAN07 |
1 |
|
SSCS24 |
Chapadão do Sul |
1 |
SSCS14 |
1 |
|
SSCS05 |
3 |
|
SSCS01 |
3 |
|
SSSM10 |
São Miguel do Passa Quatro |
6 |
SSSM12 |
1 |
|
SSSM15 |
1 |
|
SSSM03 |
1 |
|
SSSM25 |
4 |
In Silvânia-GO,
isolates from different plots on a same farm behaved differently in mycelial compatibility analyses. While three groups of mycelial compatibility were detected in the field “A”, the
25 isolates in field “B” were all compatible with each other, indicating the
existence of a single clone in this field. The existence of more than one
compatibility group in one field can be explained by the topographic position
of this field on the property. It is located in a low land area,
which contributes to a transfer of sclerotia from
higher areas by rainwater, favoring the gene flow from the population of field
“B”.
Aggressiveness of S. sclerotiorum isolates
The results of the
aggressiveness test of S. sclerotiorum isolates
are presented in Table 3. The interaction cultivar-isolate was significant
after 3 d of inoculation (P ≤
0.05), and after 7 d of inoculation (P
≤ 0.01) (Table 3). All 21 isolates used in this study could induce
disease in the cultivars BRSGO 7760 RR and M-SOY 7908 RR, with the exception of
isolate SSMO01, from Montividiu-GO, with low
aggressiveness. There was disease evolution between the first assessment, 3 d
after inoculation, and the second evaluation, 7 d after inoculation, showing
that the conditions remained favorable to the pathogen development and
expression of its aggressiveness. Regardless of the isolate, cultivar M-SOY
7908 RR was more susceptible than cultivar BRSGO 7760 RR in both evaluations,
confirming the observations under commercial field conditions.
Three days after inoculation,
the isolates SSUB18, SSSM12, SSSM03, SSCS24, and SSAF11 were the most
aggressive to cultivar BRSGO 7760 RR, causing lesions with a length range of
3.63 to 2.92 cm. The most aggressive isolates to cultivar M-SOY 7908 RR were
SSAF11, SSSM12, SSUB18, SSSM25, and SSCS24, although with no statistical
difference, causing lesion lengths of 4.11 to 3.18 cm (Table 3). Among the five
most aggressive isolates to each cultivar, four isolates (SSAF11, SSSM12,
SSUB18, and SSCS24) were common to both soybean cultivars used in this
aggressiveness analysis. Three days after inoculation, the lesion length
differed significantly between the cultivars for the isolates SSAN20, SSAN02,
SSPA11, SSM10, SSAN11, and SSSI16 and the lesion length on BRSGO 7760 RR was
shorter.
For cultivar M-SOY 7908 RR,
seven days after inoculation, the most aggressive isolates were SSSM25, SSAN20,
SSSM12, SSAN02, SSSM10, SSUB18, SSCS05, SSAF11, SSCS24, SSMO23, SSAN11, SSSI10,
SSSI37, and SSPA11. To cultivar BRSGO 7760 RR, the most aggressive isolates
were SSSM25, SSUB18, SSSM12, SSSM03, SSAF11, SSCS24, SSSI37, SSSM10, SSMO23,
SSAN20, SSSI10, and SSPA23 (Table 3). Seven days after inoculation, the number
of most aggressive isolates increased from 5 to 14 isolates; this shows that
some isolates have a longer incubation period and therefore need more time to
express their aggressiveness in soybean plants.
Among the most aggressive 14 isolates to cultivar M-SOY 7908 RR, 10 were
common to cultivar BRSGO 7760 RR. Seven days after inoculation, the difference
between cultivars in lesion length was significant for the isolates SSPA03,
SSCS05, SSPA11, SSAN 02, SSAN11, SSSI21, SSSI16, and SSUB01.Cultivar M-SOY 7908
RR proved to be the most sensitive, except for isolate SSPA03, which induced
the greatest lesion length in cultivar BRSGO 7760 RR (Table 3).
Table 3: Lesion size (cm) caused by different S. sclerotiorum isolates, representing eight groups of mycelial compatibility on the soybean cultivars BRSGO 7760
RR and M-SOY 7908 RR, three and seven days after inoculation (d.a.i.)2
Isolates |
MCG |
Cultivars (3 d.a.i.) |
Cultivars (7 d.a.i.) |
||
BRSGO 7760 RR |
M-SOY 7908 RR |
BRSGO 7760 RR |
M-SOY 7908 RR |
||
SSMO01 |
1 |
0.30 d A |
0.02 d A |
0.37 d A |
0.02 c A |
SSUB01 |
1 |
0.77 c A |
2.12 c A |
1.53 d B |
5.07 b A |
SSSI16 |
3 |
0.28 d B |
0.92 c A |
1.63 d B |
5.17 b A |
SSSI21 |
2 |
0.42 d A |
0.73 c A |
2.67 c B |
4.95 b A |
SSAN11 |
2 |
0.38 d B |
1.35 c A |
3.12 c B |
7.32 a A |
SSAN02 |
2 |
1.05 c B |
2.42 b A |
3.63 b B |
8.27 a A |
SSPA11 |
4 |
1.03 c B |
2.05 b A |
4.02 b B |
6.78 a A |
SSPA23 |
5 |
1.82 b A |
1.56 b A |
4.05 b A |
5.42 b A |
SSCS05 |
3 |
1.87 b A |
2.72 b A |
4.73 b B |
7.75 a A |
SSPA03 |
1 |
1.42 b A |
1.38 c A |
5.38 a A |
3.33 b B |
SSSI10 |
1 |
1.63 b A |
1.98 b A |
5.40 a A |
7.18 a A |
SSAN20 |
1 |
1.08 c B |
2.76 b A |
5.65 a B |
9.37 a A |
SSMO23 |
7 |
2.55 b A |
2.73 b A |
5.68 a A |
7.53 a A |
SSSM10 |
6 |
1.00 c B |
2.07 b A |
5.77 a A |
8.12 a A |
SSSI37 |
1 |
1.95 b A |
2.07 b A |
5.83 a A |
6.90 a A |
SSCS24 |
1 |
2.93 a A |
3.60 a A |
5.97 a A |
7.57 a A |
SSAF11 |
1 |
2.92 a A |
4.11 a A |
6.22 a A |
7.73 a A |
SSSM03 |
1 |
3.23 a A |
2.32 b A |
6.30 a A |
5.30 b A |
SSSM12 |
1 |
3.25 a A |
4.10 a A |
7.23 a A |
8.53 a A |
SSUB18 |
8 |
3.63 a A |
3.93 a A |
7.70 a A |
7.82 a A |
SSSM25 |
4 |
1.85 b A |
3.18 a A |
8.02 a A |
10.58 a A |
Mean |
|
1.68 2.29 |
4.80 6.70 |
||
CV (%) |
|
31.19 |
22.63 |
1Means in the original scale, but
with statistical tests resulting from the Box-Cox Power Transformation (Box and
Cox 1964)
Means followed by the same lower-case letters in the column and capital
letter in the row, did not differ from each other by the Scott-Knott test, for
the factor isolate, and Tukey test, for the factor cultivar,
at 5% significance
2Combined analysis of two experiments
Discussion
The results of the mycelial compatibility tests revealed low genetic variability
in S. sclerotiorum populations within
each sampled field (intrapopulation) at the eight
sampled municipalities, except for São Miguel do Passa
Quatro-GO, Silvânia-GO,
"A" and Patrocínio-MG, where three
compatibility groups were found, although two were represented by a only one
isolate. This indicates that the frequency of sexual recombination in these
populations, if existent, is very low. The population of Chapadão
do Sul was well-distributed in the two mycelial compatibility groups,
unlike the other populations studied, in which the groups are represented by at
most three isolates. Kolhi et al.
(1992) also concluded that there is more than one S. sclerotiorum clone in a single field, in an analysis
of 290 isolates collected in western Canada.
High genetic diversity was found in the interpopulation
analysis, i.e., analyzing the populations resulting from the mixture of
the mycelial compatibility groups of the eight
municipalities These results agree from Mahalingam et
al. (2020) based on MCG and microsatellite data which founded relatively a
high level of gene and genotypic diversity in Sri Lanka (also a tropical
country). Some researchers assert that sexual recombination occurs in regions
with a warmer climate (Atallah et al. 2004; Malvárez et al. 2007).
In Silvânia-GO, isolates from different plots
on a same farm behaved differently in mycelial
compatibility analyses. While three groups of mycelial
compatibility were detected in field "A", the 25 isolates in field
"B" were all compatible with each other, indicating the existence of
a single clone in this field. The existence of more than one compatibility
group in one field can be explained by the topographic position of this field
on the property. It is located in a lowland area, which contributes to a
transfer of sclerotia from higher areas by rainwater,
favoring the gene flow from the population of field “B”.
Among 40 isolates collected in different locations and different hosts,
five groups of mycelial compatibility and three
clusters were identified by RAPD, suggesting high genetic variability and
sexual recombination among the studied isolates (Júnior
et al. 2011). In another study, low genetic variability was observed in S.
sclerotiorum populations. Among 23 isolates
(21 isolates from common bean, 1 from potato and 1 from pepper), two
compatibility groups were observed, aside from polymorphism among isolates of a
same mycelial compatibility group (Meinhardt et al. 2002).
The intra-population genetic variability analysis was low because few compatibility
groups were detected within the sampled fields, aside from the fact that these
groups are represented by a maximum of three isolates. The only exception was
the population of Chapadão do
Sul, where the two existing compatibility groups are
distributed more homogeneously in the sampled area. A different behavior was
observed when the isolates from the different locations were paired with each
other, resulting in eight compatibility groups. In this interpopulation
analysis, group MCG1 comprised 62% of the isolates and present in all sampled
fields. The other groups (MCG2, MCG3, MCG4, MCG5, MCG6, MCG7, and MCG8)
contained at most three isolates, i.e., 9.6%. This information suggests
that the gene flow of sclerotia transferred from one
area to another through cultural practices, be it within a property or in
relatively distant areas, as well as the use of contaminated seed, contribute
little to the genetic variability of S. sclerotiorum
populations. The variability could possibly be associated with mutations,
nevertheless most clones have a low frequency, except in the population of Chapadão do Sul, where the
frequency of the two clones was similar.
Despite a few earlier studies of genetic variability in S. sclerotiorum populations in Brazil, the number of
isolates analyzed in this paper is higher than the number of isolates reported
in the literature (Gomes et al. 2011; Júnior et
al. 2011); in addition, the pathogen populations in this study were analyzed
within each sampled soybean field and between fields in different Brazilian
municipalities of Central Brazil, where white mold has become a major soybean
disease, arousing concerns of producers as well as of researchers interested in
the pathosystem S. sclerotiorum
× Glycine max L.
No relationship was observed between mycelial
compatibility groups and aggressiveness of isolates to soybean plants, since in
a same mycelial compatibility group the isolates
differed in lesion length on the stem. These results are in agreement with
those of Atallah et al. (2004), Auclair et al. (2004) and Kull et al. (2004),
who found that the MCG or microsatellite markers were not associated with
specific characteristics of aggressiveness or ecological adaptations of the
pathogen. Not all isolates of group MCG1 were equally aggressive to soybean
plants. These results disagree with those of Otto-Hanson et al. (2011),
who reported the existence of 64 mycelial
compatibility groups in a population of 156 isolates collected in different
regions of North America and France, where isolates of the same compatibility
group did not differ in aggressiveness in common bean plants Kull et al.
(2004) found 42 mycelial compatibility groups among
299 isolates from Diverse, DeKalb, Watseka Sets of Canada and Argentina. These
compatibility groups differed in aggressiveness to soybean plants. The most
aggressive were the groups with clustered isolates from different locations, e.g., Diverse, DeKalb and Watseka. These results are in agreement with
our study, since the most aggressive isolates belonging to MCG1 are from
different locations, such as São Miguel do Passa Quatro, Água Fria, Chapadão do Sul, Silvânia, Montividiu, Anápolis, and Patrocínio. It was
also found that isolates from the same location differed in aggressiveness, as in the case of the isolates SSUB 18 and SSUB 01, from Uberlândia and SSMO 01 and SSMO 23, from Montividiu, differing from the findings of Kull et al.
(2004) and Durman et al. (2003). Isolates that
were incompatible with most isolates within their respective populations (SSSM
25, SSUB 18, SSSM 10, SSMO 23, SSCS 05, SSPA 11, and SSAN 02) were included in
the group of 14 most aggressive isolates to cultivar M-SOY 7908 RR seven days
after inoculation.
The reason for the variation in aggressiveness observed in this study
may be related to the range of different municipalities in the Central region
of Brazil where sclerotia were collected, therefore
the existence of several compatibility groups would be more likely than of a
clonal population (Kolhi et al. 1992; Durman et al. 2003). Aggressiveness variation of S.
sclerotiorum has been a subject of many studies
in differents crops (Ekins et
al. 2007; Otto-Hanson et al. 2011; Attanayake
et al. 2013) also in different geographic areas.
In soybean, white mold is a severely yield-limiting disease when
favorable environmental conditions are met. Yield reductions are caused by
reduced seed number and weight (Hoffman et al. 1998; Danielson et al.
2004) resulting from the girdling of stems and disruption of xylem and phloem (Willbur et al. 2019). Thus, understanding the
diversity and aggressiveness of the pathogen is valuable to improve the
effectiveness of control practices, particularly the strategic use of cultivar
resistance. When analyzing the diversity and aggressiveness of this fungus, we
found 19 different behaviors in two soybean cultivars, thus for the selection
of resistant varieties more than one isolate should be used due to the genetic
behave in S. sclerotiorum populations.
Conclusion
Interpopulation analyses detected high genetic
diversity and eight groups of mycelial compatibility
in Central Brazil region. The S. sclerotiorum
isolates differ in terms of aggressiveness to soybean plants. This study has
found different aggressiveness levels among the 14 S. sclerotiorum isolates from soybean collected in Central
Brazil. The aggressiveness and diversity was not associated with mycelial compatibility groups.
Acknowledgements
The first author thanks CAPES,
CNPQ and FAPEG for financial support.
Author Contributions
RAG sample collection, analysis
and made the write up, VDD and VPA interpreted the results and made the write
up, RMO performed the experiments, KAGBA made the write up, MCM sample
collection and financial support, MGC sample collection, made the write up and
advisor
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