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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