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Hosseini, B.; Voegele, R.T.; Link, T.I. Fungal and Oomycete Soybean Pathogens and Molecular Detection. Encyclopedia. Available online: https://encyclopedia.pub/entry/46939 (accessed on 17 May 2024).
Hosseini B, Voegele RT, Link TI. Fungal and Oomycete Soybean Pathogens and Molecular Detection. Encyclopedia. Available at: https://encyclopedia.pub/entry/46939. Accessed May 17, 2024.
Hosseini, Behnoush, Ralf Thomas Voegele, Tobias Immanuel Link. "Fungal and Oomycete Soybean Pathogens and Molecular Detection" Encyclopedia, https://encyclopedia.pub/entry/46939 (accessed May 17, 2024).
Hosseini, B., Voegele, R.T., & Link, T.I. (2023, July 18). Fungal and Oomycete Soybean Pathogens and Molecular Detection. In Encyclopedia. https://encyclopedia.pub/entry/46939
Hosseini, Behnoush, et al. "Fungal and Oomycete Soybean Pathogens and Molecular Detection." Encyclopedia. Web. 18 July, 2023.
Fungal and Oomycete Soybean Pathogens and Molecular Detection
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This entry is adapted from the peer-reviewed paper 10.3390/jof9050587

Soybean (Glycine max) is among the most important crops. Soybean was domesticated in China over 3000 years ago and introduced to other Asian countries and, later, the Americas, Africa, and Europe. Soybean production amounted to 355,605 million tons in 2021–2022, which illustrates the enormous economic importance of this crop. Soybean is threatened by several abiotic and biotic stress factors, which result in reduction of soybean yield and quality. Pathogens, pests, and weeds cause significant losses to soybean. Important fungal and oomycete pathogens of soybean and molecular methods to detect them are presented.

seed-borne fungi soil borne pathogens molecular detection methods species

1. Genus Diaporthe

The genus Diaporthe Nitschke (1870) (asexual state Phomopsis (Sacc.) Bubák) includes hundreds of species. Diaporthe species are widespread and they are non-pathogenic endophytes (biotrophic fungi), saprotrophs, and fungal pathogens of many plants and even mammals [1][2].
Phytopathogenic Diaporthe species have been intensively studied, especially those affecting economically important crops, such as soybean, sunflower, grapes, citrus, and fruit, and ornamental trees [3]. Diseases caused by Diaporthe spp. on soybean are stem canker, pod and stem blight, and seed decay (Table 1) [4][5][6].
Morphological evaluation of fungal growth from surface sterilized soybean seeds plated on acidified potato dextrose agar (APDA) is still common in the identification of Diaporthe spp. [7]. However, because of strong similarities and overlaps in shapes and colors of cultures and in conidial size, delimitation of Diaporthe spp. is not valid just based on morphology [1][2][8]. Species diversity in the genus Diaporthe was explored by assays using PCR [8][9][10]. The nuclear ribosomal internal transcribed spacer (ITS) can be used for discrimination of Diaporthe spp. [11][12][13]. The primers Phom.I and Phom.II were designed on the ITS sequences of D. phaseolorum and D. longicolla for the detection of many Diaporthe spp. [9]. The primers DphLe and DphRi were developed for detection of D. aspalathi [14]. There are also real-time (q)PCR assays based on ITS to detect and quantify Diaporthe spp. on soybean. The first was developed by Zhang et al. [15]. The TaqMan primer-probe sets PL-3, PL-5, and DPC-3 were designed for D. longicolla, D. aspalathi, D. caulivora, and D. sojae. For additional information on the mentioned primers and assays, see Table 1.
Table 1. Primers and corresponding assays for the diagnosis of Diaporthe spp. on soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Assay Ref.
ITS Diaporthe sp., but based on D. phaseolorum and D. longicolla Phom.I GAGCTCGCCACTAGATTTCAGGG 60 PCR [9]
Phom.II GGCGGCCAACCAAACTCTTGT
ITS D. aspalathi DphLe TCGGCCTTGGAAGTAGAAGAC 60 PCR [14]
DphRi ACTGAATGCGTTGCGATTCT
ITS D. caulivora DPC-3F TTTATGTTTATTTCTCAGAGTTTCAGTGTAA 60 qPCR [15]
DPC-3R GGCGCACCCAGAAACC
DPC-3P FAM-CGGGCTGCTCCCCGTCTCC-TAMRA
ITS D. longicolla PL-3F CAGAGATTCACTGTAGAAACAAGAGTTT 60 qPCR [15]
PL-3R CCGGCCTTTTGTGACAAA
PL-3P FAM-CGGGCTGCTCCCTGTCTCCAG-TAMRA
ITS D. longicolla, D. aspalathi, D. sojae PL-5F CGAGCTCGCCACTAGATTTCA 60 qPCR [15]
PL-5R CCTCAAGCCTGGCTTGGTGATGG
PL-5P FAM-CCATCACCAAGCCAGGCTTGAGG-TAMRA
TEF D. longicolla DPCL-F TGTCGCACCTTTACCACTG 60 qPCR a [16]
DPCL-R GAACGATCCAAAAAGCTCTC
DPCL-P FAM-GCATCACTTTCATTCCCACTTTCTG-BMN-Q535
TEF D. caulivora DPCC-F GCCTGCAAAACCCTGTTAC 60 qPCR a [16]
DPCC-R CATCATGCTTTAAAAATGGGG
DPCC-P Cy5-CTCTTACCACACCTGCCGTCG-BMN-Q620
TEF D. eres DPCE-F ACTCACTCAATCCTTGTCAC 60 qPCR a [16]
DPCE-R GAGGGTCAGCATAATATTCG
DPCE-P ROX-CCATCAACCCCATCGCCTCTTTC-BMN-Q590
TEF D. novem DPCN-F AAAACCCTGCTGGCATTAAC 60 qPCR a [16]
DPCN-R TATTCTTGACAGTTCGTTTCG
DPCN-P HEX-TCTACCACTTTCAACCCTATCAATC-BMN-BMN-Q535
a Probe based real-time PCR. These four primer-probe-combination were designed for use together in a quadruplex reaction.
However, ITS sequence data alone are not sufficient to resolve all Diaporthe species [17][18]. Therefore, translation elongation factor 1-α (TEF1), beta-tubulin (TUB), calmoduline (CAL), histone-3 (HIS), and large ribosomal subunit (LSU) are also used to differentiate Diaporthe species [19][20][21].
Recently, the TaqMan primer-probe sets DPCL, DPCC, DPCE, and DPCN were designed based on TEF1 to identify, discriminate, and quantify D. longicolla, D. caulivora, D. eres, and D. novem simultaneously in a quadruplex real-time PCR [16].
In a study on expression of soybean defense-related genes, the TUB-based primers DPM were used with SYBR® Green real-time PCR to detect and quantify D. aspalathi in soybean tissues [22]. The same assay was also used to quantify the fungal biomass in soybean stems infected with D. caulivora [23].
There are additional PCR assays for the identification of Diaporthe spp.: random amplified polymorphic DNA (RAPD), PCR-restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) have been used to distinguish Diaporthe species [9][10][24][25][26]. The species D. caulivora, D. aspalathi, and D. sojae could only be defined after finding differences between the D. phaseolorum varieties caulivora, meridionalis, and sojae using RAPD [24]. After that, PCR-RFLP was used to distinguish D. longicolla, D. caulivora, D. aspalathi, and D. sojae [9].

2. Genus Sclerotinia

Sclerotinia sclerotiorum (Lib.) de Bary is among the most destructive plant pathogens. It is the only relevant pathogen of the genus Sclerotinia on soybean. This fungus can infect over 400 species of plants, including sunflower, soybean, and oilseed rape [27]. On soybean S. sclerotiorum causes white mold, which reduces yield by more than 40% in wet and mild weather [28]. Since the pathogen disseminates via seeds, sowing seeds with certified health quality is the first step to avoid this disease.
Molecular detection has been established independently more than once. Primers SSFWD/SSREV were designed for the identification of S. sclerotiorum [29] and the specificity and sensitivity of these primers were confirmed experimentally by performing PCR and real-time (q)PCR (SYBR Green) using the touchdown method, to distinguish S. sclerotiorum isolates from Aspergillus, Cercospora, Colletotrichum, Corynespora, Fusarium, Macrophomina, Diaporthe, and Botrytis species [30]. Other primers were used to identify S. sclerotiorum ascospores and detect S. sclerotiorum in infected plant tissue [31][32][33]. When tested on soybean seeds inoculated with S. sclerotiorum, these primers were not useful [30]. Variability among isolates from this species from different regions or hosts can lead to these differences. In a separate study, the primer pair SSFWD/SSREV [29] was tested to detect S. sclerotiorum in inoculated and in naturally infected soybean seeds using the seed soaking procedure [28]. Since the ITS region did not allow for fully reliable diagnosis the mitochondrial small rRNA and SS1G_00263, coding for a hypothetical secreted protein was used [33][34]. For additional information on the mentioned primers and assays, see Table 2.
Table 2. Primers and corresponding assays for diagnosis of Sclerotinia sclerotiorum on soybean.
Target Gene Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS SSFWD GCTGCTCTTCGGGGCCTTGTATGC 65 a 278 PCR and qPCR [29]
SSREV TGACATGGACTCAATACCAAGCTG
ITS M13FWD GTAAAACGACGGCCAGT   252 qPCR b [31]
M13REV CAGGAAACAGCTATGAC
mitochodrial small rRNA mtSSFor AGGTAACAAGTCAGAAGATGATCGAAAGAGTT   125/80? qPCR [33]
mtSSRev GCATTAAGCCTGTCCCTAAAAACAAGG
SS1G_00263 SSBZF GCTCCAGCAGCCATGGAA 60   qPCR [34]
SSBZR TGTTGAAGCAGTTGACGAGGTAGT
SSBZP CAGCGCCTCAAGC
a Used in touchdown PCR. b Eva Green based real-time PCR. These four primer pairs can be used together in two duplex reactions where the products are distinguished by their melting temperature.

3. Genus Colletotrichum

The ascomycete genus Colletotrichum is quite large, with more than 200 species. Some of the species are clearly defined, but there also are several species complexes [35][36][37]. Colletotrichum spp. are causal agents of anthracnose in more than 3000 plant species and are among the top 10 fungal pathogens [38][39][40]. C. truncatum, C. destructivum, C. coccodes, C. chlorophyte, C. gloeosporioides, C. incanum, C. plurivorum, C. sojae, C. musicola, and C. brevisporum can all cause anthracnose on soybean [36][41][42][43][44][45][46][47][48]. Among those species C. truncatum is the most notorious [41] and there is relatively little information about the other species infecting soybean. Phylogenetic resolution is still in progress, with many studies addressing the issue [35][36][37][38][49][50][51][52][53][54].
Variation in the ITS region is not sufficient to discriminate all species, so Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), TUB2, CHS-1, and ACT are used along with ITS, also when using with the soybean pathogens [36][49]. Among those GAPDH is most informative, at least for distinguishing the most important pathogen C. truncatum [55]. Based on ITS, three primers were designed and used to distinguish C. gloeosporioides and C. truncatum on soybean by performing classical multiplex-PCR [56]. The intergenic spacer (IGS) has been used as an alternative to ITS. An advantage is that it contains more polymorphic sites. It was efficiently used for detecting C. lupini in lupins by PCR and can be considered an alternative target for Colletotrichum species [57].
A multiplex TaqMan qPCR assay targeting the GAPDH gene was developed to detect and quantify C. truncatum along with Corynespora cassiicola and S. sclerotiorum in soybean seeds [58]. A multiplex qPCR assay targeting the cox1 gene has also been established to distinguish the four soybean-infecting Colletotrichum species C. chlorophyti, Glomerella glycines (Colletotrichum sp.), C. incanum, and C. truncatum, by using two duplex sets. Successful detection was achieved with 0.1 pg of C. truncatum DNA, but when published, the assay had not yet been tested on host tissue samples [59].
For some Colletotrichum species that can infect soybean, diagnostic assays were developed because of damages on other host plants. These are C. acutatum on strawberries and grapevines [60][61], C. coccodes in soil and on potato tubers [62], C. kahawae on coffee [63], and C. lagenarium on cucurbit crops [64]. These assays may be transferred and used for diagnostics on soybean, too.
In addition to PCR and real-time PCR also LAMP (loop-mediated isothermal amplification) assays were established to detect C. truncatum, targeting the large subunit of RNA polymerase II (Rpb1) coding gene [65], and C. gloeosporioides, for which the target gene was a glutamine synthetase (GS) [66]. While these assays offer the advantage of diagnosis directly on the field because no PCR cycler is necessary and the reaction can be observed directly without any equipment, they are an order of magnitude less sensitive than the corresponding real-time PCR assays. For additional information on the mentioned primers and assays, see Table 3.
Table 3. Primers and corresponding assays for diagnosis of Colletotrichum spp. On soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
cox1 C. chlorophyti cox1AF CCTGGTATAAGATTACATAAG 55 115 qPCR a [59]
cox1AR CTGTAAGTACCATAGTAATTG
cox1 C. sojae cox18AF ACATTTATCAGGAGTAAGTAG 55 77 qPCR a [59]
cox18AR TTCCAGGTGTTCTCATAT
cox1 C. incanum cox6AF-2 ATGAACATTATATCCTCCTT 55 115 qPCR a [59]
cox6AR-2 ATTAACTGCTCCTAATAAAC
cox1 C. truncatum cox15BF TTATGCCAGCCTTAATAG 55 117 qPCR a [59]
cox15BR AAGATGGTGGTAATAATCA
ITS C. gloeosporioides Colg 1 AACCCTTTGTGAACATACC 63 443 qPCR [56]
Colg 2 CCCTCCGGATCCCAG
ITS C. truncatum Colg 1 AACCCTTTGTGAACATACC 63 375 qPCR a [56]
CT 2 CTTTAAGGGCCTACGTCAA
ITS C. acutatum CaITS_F701 GGATCATTACTGAGTTACCGC 60 80 qPCR [60]
CaITS_R699 GCCCGCGAGAGGCTTC
CaITS_R815 b GCCCACGAGAGGCTTC
CaITS_P710 TACCTAACCGTTGCTTCGGCGGG
ITS C. acutatum ACUT-F1 CGGAGGAAACCAAACTCTATTTACA 60 70 qPCR [61]
ACUT-R1 CCAGAACCAAGAGATCCGTTG
ACUT-PB CGTCTCTTCTGAGTGGCACAAGCA
ITS C. gloeosporioides GLOE-F1 GGCGGGTAGGGTCYCCG 60 101 qPCR [61]
GLOE-R2 ACTCAGAAGAAACGTCGTTAAATCAG
GLOE-PB CTCCCGGCCTCCCGCCYC
ITS Colletotrichum sp. COL GEN-F1 TGCCTGTTCGAGCGTCATT 60 111 qPCR [61]
COL GEN-R2 CTACGCAAAGGAGGCTCCG
COL GEN-PB AACCCTCAAGCWCYGCTTGGYKTTGG
IGS C. lupini CLF CCCGAGAAGGCTCCAAGTA 63   PCR [57]
CLR CATAAACGCCTAAGAACCGC
GAPDH C. truncatum ColT-F6 TTGAGACCAAGTACGCTGTATGTATCAC 60   qPCR [58]
ColT-R5 TTCTGCCTCACATCGAACTCTC  
ColT-P HEX-CAGCCTTCG/ZEN/ACTCTCGTTGGAAAA-IABkFQ  
GS C. gloeosporioides F3 GCTGCAGCCGGAAAATCC 64   LAMP [66]
B3 GGCAGACTCGGAGAGACC
FIP (F1c + F2) ACCGGCTCAGCTGCAACGC-ACACGAGCAAAAGGATACGC
BIP (B1c + B2) TAATGCCTTTCACGACCTGCGG-CCGAGGCAATGATTCCTCAA
LF CGGGCCAACGCTGGAAAA
LB GGCGCAACAAAGCTGGG
Rpb1 C. truncatum F3 ACGGAGAATACTCTCTGGGT 62   LAMP [65]
B3 AGGATGTTGTGTGCCATCTC
FIP (F1c + F2) GCCTTGTGTCGGACTCTGGG-GCAAGCTCCCGTTAACCA
BIP (B1c + B2) ACAGCTTGTCGCCAAGTACGAG-GGGTGTGATCTGAGGCTCTT
LF TGAATGTTGCCACAGCCGC
a Eva Green based real-time PCR. These four primer pairs can be used together in two duplex reactions where the products are distinguished by their melting temperature. b Alternative reverse primer to cover intraspecific sequence variation.

4. Genus Fusarium

Multiple Fusarium species are among the most important phytopathogenic and mycotoxigenic fungi [67][68]. Several Fusarium species are associated with soybean, causing Fusarium blight/wilt (F. oxysporum), sudden death syndrome (SDS, F. virgiliforme formerly F. solani f. sp. glycines), and root rot and seedling diseases (several Fusarium spp.) [69][70][71][72][73][74][75]. Diagnosis of the root pathogens may be difficult because they may either be the primary pathogen or infect together with other soilborne fungi (e.g., Macrophomina, Phytophthora, Pythium, and Rhizoctonia) [71][76]. Fusarium species have some differences in their housekeeping genes, and molecular identification has been widely used. Relevant genes for this genus are TEF1, TUB, mitochondrial small subunit rDNA (mtSSU), 28S rDNA, ITS, and IGS [77][78][79][80].
The causal agent of SDS, F. solani (Mart.) Sacc. f. sp. glycines [81], was first identified in 1989 [82][83]. At first, its identification has relied on morphological characteristics, which did not fully resolve the species complex. Using nuclear ribosomal DNA sequences of species within the F. solani complex, SDS-causing isolates were identified and F. solani f. sp. phaseoli was defined [77]. However, non-SDS-causing isolates are still included within F. solani f. sp. phaseoli. Another study using RAPD, could show that SDS-causing isolates form a cluster representing a biological subgroup within F. solani f. sp. phaseoli, and the authors suggested that it represents a separate forma specialis [84]. The differences between F. solani f. sp. glycines causing SDS and other F. solani are important for specific identification and detection. Differences in the mtSSU rRNA gene can be used to distinguish isolates of F. solani [78]. Detection of F. solani f. sp. glycines from plant and soil samples was enabled by a PCR assay using primers based on mtSSU and TEF1 [78][85].
Two TaqMan probe qPCR assays for quantification of F. virguliforme from soybean plant samples based on mtSSU sequences are available [86][87]. Due to similarities in the F. solani species complex, the mitochondrial DNA (mtDNA) region is too conserved to differentiate F. virguliforme from the dry bean root rot pathogens F. cuneirostrum and F. phaseoli and other SDS causal agents, such as F. tucumaniae, F. crassistipitatum, and F. brasiliense, which dominate in South America [88][89]. However, the IGS region of the rDNA can resolve F. virguliforme from the other closely related Fusarium species, as demonstrated in multilocus genotyping studies of clade 2 F. solani species [88][89]. Following this, a TaqMan primer/probe set based on IGS to detect and quantify F. virguliforme in field-grown soybean roots and soil was established [90]. The FvTox1 gene was also used to distinguish F. virguliforme from the other species within the SDS-BRR (bean root rot) clade in soil and soybean root samples with a species-specific TaqMan real-time qPCR assay [91].
Since the assay based on the FvTox1 gene has a much higher limit of detection than assays based on the rDNA, yet another assay specific for F. virguliforme was developed, based on the IGS region [92]. This primer/probe set was later also used in a duplex qPCR assay for simultaneous detection of F. virguliforme and F. brasiliense [93]. Most recent (to our knowledge, 2022) is a series of primer/probe sets to detect F. acuminatum, F. graminearum, F. proliferatum, and F. solani, based on TEF1 and F. oxysporum, and F. equiseti, based on IGS [94]. In this case, there are limits in specificity, especially of the F. solani primers/probe set (Fsol), which also amplifies F. graminearum, F. equiseti, and F. virguliforme, though much later than F. solani. For additional information on the mentioned primers and assays, see Table 4.
Table 4. Primers and corresponding assays for diagnosis of Fusarium spp. on soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS1 F. solani f. sp. phaseoli FspF ACCCCCTAACTCTTGTTATATCC 60 957–958 PCR [77]
FspR GCGCAATACCCTGAGGCG
TEF1 F. solani f. sp. phaseoli Effp-1 AACCCCGCCCGAGGACTCA 72 562 PCR/qPCR [79]
Effp-2 AGACATGAGCGATGAGAGGCA
TEF1 F. solani f. sp. glycines FsgEF1 GAGTCGGTTAGCTTCTGTC 66 a/56 237 PCR b [85]
FsgEF2 GCGCGCCTTGCTATTCTCC
mtSSU F. solani f. sp. glycines Fsg1 GTCTTCTAGGATGGGCTGGT 66 b/56 438 PCR c [85]
Fsg2 CATTTAATGCCTAGTCCCCTATCA
mtSSU F. solani f. sp. glycines Fsg-q-1F GATACCCAAGTAGTCTTTGCAGTAAATG 60   qPCR [86]
Fsg-q-1R TTAATGCCTAGTCCCCTATCAACAT
Fsg-q-1P 6FAM-TGAATGCCATAGGTCAGAT-MGBNFQ
mtSSU F. solani f. sp. glycines FSGq1 AACCCTTTGTGAACATACC 60   qPCR [87]
FSGq2 CCCTCCGGATCCCAG
FSG-MGB probe 6FAM-TCTTCTAGGATGGGCTGGT-MGBNFQ
FvTox1 F. virguliforme FV-F GCAGGCCATGTTGGTTCTGTA 60 200 qPCR [91]
FV-R GCACGTAAAGTGAGTCGTCTCATC
FV-MGB probe 6FAM-ACTCAGCGCCCAGGA-MGBNFQ
IGS F. virguliforme FvIGS-F1 GGTGGTGCGGAAGGTCT 66   qPCR [90]
FvIGS-R3 CCCTACACCTTTCGTACCAT
FvIGS-Probe2 6FAM-ATAGGGTAGGCGGATCTGACTTGGCG-TAMRA
IGS F. virguliforme F6-3 GTAAGTGAGATTTAGTCTAGGGTAGGTGAC 60   qPCR [92]
R6 GGGACCACCTACCCTACACCTACT
FvPrb-3 6FAM-TTTGGTCTAGGGTAGGCCG-MGBNFQ
IGS F. brasiliense Fb_F2 AGGTCAGATTTGGTATAGGGTAGGTGAGA 67 f 130 qPCR d [93]
Fb_R2 CGGACCATCCGTCTGGGAATTT 66 f
Fb_Prb1 5HEX-TGGGATGCCCT+AATTTTT+ACGG-3IABkFQ e 65 f
TEF1 F. acuminatum FacuF TCGCGCACTACATGTCTT 54 g 142 qPCR [94]
FacuR AGAGAGCGATATCAATGGTGA 53 g
FacuP FAM-AACCACTGG/ZEN/ACAATAGGAAGCCGC 61 g
TEF1 F. graminearum FgraF CTCTTCCCACAAACCATTCC 53 g 104 qPCR [94]
FgraR TACTTGAAGGAACCCTTACC 51 g
FgraP FAM-ACCACCTGT/ZEN/CAATAGGAAGCCGCC 63 g
TEF1 F. proliferatum FproF GCGTTTTTGCCCTTTCCTGT 57 g 123 qPCR [94]
FproR AACCCAGGCGTACTTGAAGG 57 g
FproP FAM-AGGAAGCCG/ZEN/CTGAGCTCGGT 64 g
TEF1 F. solani FsolF AAACCCTCATCGCGATCTG 55 g 108 qPCE [94]
FsolR AGTGACCGGTCTGTAGATGA 55 g
FsolP FAM-CCTGGTATC/ZEN/TCGGGCGGG 60 g
IGS F. equiseti FequiF TGTTGGGACTCGCGGTAA 56 g 94 qPCR [94]
FequiR GATTACCAGTAACGAGGTGTA 51 g
FequiP FAM-CACGTCGAG/ZEN/CTTCCATAGCGTAGT 60 g
IGS F. oxysporum FoxyF CCGTCGATAGGAGTTCCGTC 57 80 qPCR [94]
FoxyR TCGAACCGACCATCTCCAAG 57
FoxyP FAM-TGGACGGTG/ZEN/CAGGGTAGG 64
a First is the first melting temperature in the touchdown program, the other is the last. b For optimal specificity, a protocol for touchdown PCR is used. For optimal sensitivity, a nested PCR is described using additional general TEF1 primers for the first round. c For optimal specificity, a protocol for touchdown PCR is used. For optimal sensitivity, a nested PCR is described using additional general mtDNA primers (NMS) for the first round. d The assay was designed for duplexing with the primer/probe set for F. virguliforme above. e The + sign indicates that the next base is a locked nucleic acid (LNA) residue. These are necessary because the probe covers only two nucleotides that differ between F. brasiliense and other Fusarium spp., i.e., F. virguliforme. Do not be discouraged when checking Figure 1 in [93]; there the probe sequence is given wrongly and the position is off by one base, but the sequence given here is correct. f Calculated primer melting temperatures. The actual annealing temperature used was not reported. g Calculated primer melting temperatures. All these assays were run with an annealing temperature of 60 °C.

5. Genus Cercospora

Two species of genus Cercospora can infect soybeans. C. kikuchii (T. Matsumoto and Tomoy; M. W. Gardner) causes Cercospora leaf blight (CLB) and purple seed stain (PSS), while Frogeye leaf spot is caused by C. sojina Hara [69][95][96]. Production of a red toxin called cercosporin by C. kikuchii is recognized as a pathogenicity factor during colonization of soybean seeds and other aerial parts of the plant, including leaves, petioles, stems, and pods [97][98][99]. Cercosporin is also responsible for the symptoms of PSS: presence of purple spots against the natural color of the soybean seed coat [97] and causes membrane damage and cell death [100]. Cercosporin production is regulated by CFP (Cercosporin Facilitator Protein), which is specific for the genus and encoded by the gene cfp [101].
Seven nuclear gene regions and the mitochondrial (cyb) gene region were evaluated to study Cercospora species phylogenetically [102]. The seven regions included ACT, CAL, HIS, ITS, and TEF1, which were used in previous studies [103][104][105][106]. In addition, two primer pairs were designed based on cfp of C. kikuchii and one new primer Ck_Betatub-F1 to amplify tub-1 after the complete TUB from C. beticola was analyzed [102]. TUB and cfp are excellent sources for polymorphic markers to investigate the relationships between the CLB and PSS pathogens.
The CNCTB6F/CNCTB6F primer pair, which targets the NADPH-dependent oxidoreductase gene (CTB6) from C. nicotianae [107], can be used to detect C. kikuchii and to differentiate between C. kikuchii and C. sojina [108]. The ITS1 and ITS2 sequences of genus Cercospora are too similar to develop primers for species-specific detection [108]. Therefore, a TaqMan real-time PCR assay was developed based on the CTB6 gene to detect C. kikuchii [108] (Table 5).
Table 5. Primers and corresponding assays for diagnosis of Cercospora kikuchii on soybean.
Target Gene Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Reference
CTB6 CKCTB6-2F CACCATGCTAGATGTGACGACA     qPCR [108]
CKCTB6-2R GGTCCTGGAGGCAGCCA
CKCTB6-PRB CTCGTCGCACAGTCCCGCTTCG 

6. Genus Septoria

Septoria glycines Hemmi causes Septoria brown spot, a foliar disease on soybean [69]. The pathogen infects pods and seeds but is rarely transmitted by seeds [109]. Early in the season, the symptoms are similar to those of bacterial blight (Pseudomonas syringae pv. glycinea) [110][111]. Later in the season, Septoria brown spot occurs together with frogeye leaf spot (Cercospora sojina) and Cercospora leaf blight (C. kikuchii) [112], making molecular diagnosis especially useful.
Three primers/probe sets were developed for qPCR based on ACT, TUB, and CAL [113]. The CAL set was not as specific as anticipated. The ACT set (Table 6) was specific to S. glycines for both conventional PCR and qPCR and the TUB set was specific only in qPCR.
Table 6. Primers and corresponding assay for diagnosis of S. glycines on soybean.
Target Gene Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Reference
ACT Ac1(f) ACAATCCAGGGACCACAATC 60 a 90–100 qPCR [113]
Ac2(r) ATGGCTGATCGCATACCC 59 a
Ac(probe) 6FAM-AGAGCTGACCAGGACCCAGCATCCA-TAM 73 a
a Calculated primer melting temperatures. The assay was run with an annealing temperature of 60 °C.

7. Genus Macrophomina

The species in genus Macrophomina that is relevant to soybean is Macrophomina phaseolina (Tassi) Goid. This is one of the most severe soil and seed borne pathogens, attacking a wide range of hosts [114]. The fungus causes damping off, seedling blight, collar rot, stem rot, charcoal rot, and root rot diseases in various crops [115]. In soybean, M. phaseolina causes charcoal rot.
There is sequence variation between isolates of M. phaseolina. There have been several attempts to correlate this variation in several genetic markers with sampling region and host plant association, but while some groups found correlations [116], other publications could not [117][118][119], so that no forma speciales for soybean or another host plant have been defined, yet.
Consequently, the molecular detection assays that were established so far aim to detect all strains of M. phaseolina. In one approach that targets the ITS, the authors made sure to find primers on regions in the ITS that are conserved among M. phaseolina isolates, but different from other species [120]. The other approach to find a sequence conserved among M. phaseolina isolates was the sequence characterized amplified regions (SCAR) method. Using the universal rice primer URP-9F, a PCR product could be obtained that was the same for all M. phaseolina isolates and the resulting sequence (gene of unknown function) was used to design primers and probe for qPCR, enabling either SYBR green based or probe based qPCR [121]. A LAMP assay for detection of the species also uses the ITS sequence [122]. For additional information on the mentioned primers and assays, see Table 7.
Table 7. Primers and corresponding assays for diagnosis of M. phaseolia.
Target Gene Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS MpKFI CCGCCAGAGGACTATCAAAC 56 350 PCR [120]
MpKRI CGTCCGAAGCGAGGTGTATT
MpKH1 a GCTCTGCTTGGTATTGGGC 55 a   dot blot
Ukn b MpSyK F ATCCTGTCGGACTGTTCCAG 60   qPCR [121]
MpSyK R CTGTCGGAGAAACCGAAGAC  
MpTqK F GCCTTACAAGGGTCTCGTCAT 60   qPCR
MpTqK R CCCTTGGCGATGCCGATA  
MpTqK P 6-FAM-CAGGCCACAGGATCTT-MGBNFQ  
ITS F3 GCACATTGCGCCCCTTG 62 c   LAMP [122]
B3 GTTCAGAAGGTTCGTCCGG  
FIP AGGACGGTGCCCAATACCAAGCGGGGCATGCCTGTTCGA  
BIP CTCAAAGACCTCGGCGGTGGGCTCCGAAGCGAGGTGTA  
a DIG labeled probe. Correspondingly also the temperature is the hybridization temperature. b Sequence obtained using the SCAR approach. c Incubation for the LAMP assay. Incubation for 60 min.

8. Genus Phialophora

Brown stem rot (BSR) is a vascular disease caused by the soil-borne fungus Phialophora gregata f. sp. sojae (Allington and Chamberlain) Gams. This pathogen has two genotypes, “A” and “B” [123]. Genotypic differences among isolates correspond to phenotypic differences in the type and severity of symptoms. Isolates of genotype “A” are more aggressive than isolates of genotype “B” [124][125]. Genotypes “A” and “B” differ by a 188-bp insertion/deletion (INDEL) in the IGS of the ribosomal DNA and they also display cultivar preference [126][127][128].
Primers based on ITS were developed to identify P. gregata in infected soybean stems [129]. This primer pair was also used in combination with primer pair Plect1/Plect2 specific for Plectosporium tabacinum, to differentiate these two pathogens, which are associated with BSR [130]. Once the IGS region was found useful to distinguish “A” and “B”, primers BSRIGS1 and BSRIGS2 were designed [126]. In 2007, a qPCR assay was developed to quantify P. gregata f. sp. Sojae in plant tissue and in soil [131]. This qPCR assay does not yet distinguish between genotypes “A” and “B”. In 2009, a qPCR to specifically detect genotype “A” was developed. In combination with a specific primer/probe set [131], genotype “B” can also be quantified by determining the difference between the total P. gregata f. sp. sojae DNA amount and that of genotype “A” [132]. For additional information on the mentioned primers and assays, see Table 8.
Table 8. Primers and corresponding assays for diagnosis of P. gregata f. sp. sojae.
Target Gene Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS BSR1 GCTTGCTCCGTGGCGGGCTG 60 480 PCR [129]
BSR2 AATTTGGGTGTTGCTGGCATG
IGS BSRIGS1 GGGGTTCCGGGATTCACAGG 55 1020 a PCR [126]
BSRIGS2 GAGTGGTAAATGGGGTAATCAAC 830 a
IGS BSRqPCRf1 CAAACCAGGGCCGATCAG 60   qPCR [131]
BSRqPCRr1 CGGATTCAGCGTAAAAAATGG  
BSRqPCRpb1 6-FAM-CTCCCGTATGGTTTCT-MGBNFQ  
IGS b PgsAspF GGAATTGGTGGGAGAGG 60 69 qPCR [132]
PgsAspR GACTTCTAGGGTATGTCTACAGTG
PgsAspPR CAL Flour Red 610-AGGCTACTCTTACAGGCTCTC-BHQ-2
a The PCR product is 1020 bp for genotype A and 830 bp for genotype B. This is how this assay can distinguish the two genotypes. b Target in this special case is the INDEL sequence specific for genotype A. It is impossible to get primers specific for genotype B using the same strategy, which is why for quantification of genotype B these primers are used together with the assay by [131].

9. Genus Rhizoctonia

Rhizoctonia solani Kühn (Teleomorph: Thanatephorus cucumeris (Frank) Donk) is a soil-borne fungal pathogen. R. solani is a species complex that was subdivided into anastomosis groups (AGs) based on an assessment of hyphal fusions [133][134]. AGs are subdivided into subgroups based on cultural morphology and physiological characteristics [135]. There are “13 AGs with 14 subgroups” [135][136][137]. The AGs vary in morphology, pathogenicity, and susceptibility to fungicides [138]. AG 1 to 4 cause disease in several economically important crops [133]. The other groups have more restricted host ranges or are less important. AG 12 is a special case: it forms mycorrhiza [136]. Molecular phylogenies have confirmed the AGs and the nomenclature was kept, even though the AGs may represent different species. The AGs important for soybean are AG 1-IA and AG 1-IB [69].
PCR detection assays were established for most of the AGs. Here, researchers present the assays for AG 1-IA and AG 1-IB. An assay based on ITS to detect AG 1-IA, AG 1-IB, and other AGs was designed in 2002 [139]. Later, two other groups [140][141] reported additional assays for the two AGs, respectively. As part of a real-time PCR study to detect and discriminate 11 AGs of R. solani using ITS regions, AG 1-IA was also detected [142]. In 2015, a LAMP assay was developed to detect R. solani in infected soybean tissues in the field [122]. For additional information on the mentioned primers and assays, see Table 9.
Table 9. Primers and corresponding assays for diagnosis of R. solani on soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
28S ribosomal DNA R. solani AG-common f CTCAAACAGGCATGCTC 54   PCR [139]
AG 1-IA AG 1-IA CAGCAATAGTTGGTGGA 265
AG 1-IB AG 1-IB AAGGTCCTTTGGGGTTGGGG 300
Unk a AG 1-IB N18-rev AGCGTGCTAACATAGTCACTC   324 PCR [140]
N18-for ACACTAGAGTAGGTGGTATCA
ITS AG 1-IA Rs1F GCCTTTTCTACCTTAATTTGGCAG 60 137–140 qPCR [141]
Rs2R GTGTGTAAATTAAGTAGACAGCAAATG
ITS1 AG 1-IA AG-1-1A_F TTGTTGCTGGCCTTTTCTACCT 60   qPCR [142]
AG-1-1A_R ATGGAATTAAATCCACCAACTATTGCT  
AG-1-1A_P FAM-CATCACACCCCCTGTGCACTTGTGAGA-TAMRA  
ITS R. solani F3 CGAAATGCGATAAGTAATGTGAA 62 b   LAMP [122]
B3 AGAGGAGCAGGTGTGAAG
FIP GCTCCAAGGAATACCAAGGAGCCAGAATTCAGTGAATCATCGAATC
BIP TGCCTGTTTGAGTATCATGAATTCTAAAAGACCTCCAATACCAAAG
a Sequence obtained by sequencing a RAPD fragment. SCAR method for primer identification. b Incubation for the LAMP assay. Incubation for 60 min.

10. Genus Phakopsora

Soybean rust (SBR) is considered the economically most important disease on soybean. SBR is caused by two closely related fungi, Phakopsora pachyrhizi and P. meibomiae [143][144]. P. pachyrhizi, also called Asian soybean rust (ASR) since it originates from East Asia, is more aggressive and causes considerably greater yield loss [144]. The two species may be confused and early symptoms can be confused with bacterial pustule [69]. Therefore, molecular diagnosis of the soybean rust has been established. The ITS region has more than 99% nucleotide sequence similarity among different isolates of either P. pachyrhizi or P. meibomiae, but only 80% between the two species. Using the differences within the ITS region, four sets of primers were designed for P. pachyrhizi (Ppa1/Ppa2, Ppa3/Ppa4, Ppm1/Ppa2, and Ppm1/Ppa4) and two sets for P. meibomiae (Pme1/Pme2 and Ppm1/Pme2). The primers were tested and Ppm1/Ppa2 used as specific for P. pachyrhizi and Ppm1/Pme2 for P. meibomiae [145]. A VIC-labeled probe and the primers Ppm1/Ppm2 were designed as specific for genus Phakopsora.
P. pachyrhizi urediospores are wind-dispersed and, apart from diagnosis on plants, detection of spores in the air can be useful to predict epidemics or for scouting efforts. For this, the assay described above and another assay [146] were tested for sensitivity and from these assays another nested assay was created with a newly designed TaqMan probe (ITS1PhpFAM1). The nested assay combines the reverse primer Ppa2 specific to P. pachyrhizi [145] and a more general rust fungal forward primer ITS1rustF4a in the first round. In the second round ITS1rustF10d and ITS1rustR3d are combined with the probe. The assay can detect single and so is sensitive enough to find spores deposited in rain [147]. For additional information on the mentioned primers and assays, see Table 10.
Table 10. Primers and corresponding assays for diagnosis of P. pachyrhizi and P. meibomiae on soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS P. pachyrhizi Ppm1 GCAGAATTCAGTGAATCATCAAG 65 a 60 b 141 PCR/qPCR [145]
Ppa2 GCAACACTCAAAATCCAACAAT
ITS P. meibomiae Ppm1 GCAGAATTCAGTGAATCATCAAG 139
Pme2 GCACTCAAAATCCAACATGC
ITS Phakopsora Ppm1 GCAGAATTCAGTGAATCATCAAG 77
Ppm2 CTCAAACAGGTGTACCTTTTGG
ITS Phakopsora FAM-probe c FAM-CCAAAAGGTACACCTGTTTGAGTGTCA-TAMRA  
VIC-probe d VIC-TGAACGCACCTTGCACCTTTTGGT-TAMRA  
ITS P. pachyrhizi ITS1rustF4a e GAGGAAGTAAAAGTCGTAACAAGGTTTC 60   nested qPCR [146][147]
ITS1rustF10d TGAACCTGCAGAAGGATCATTA
ITS1rustR3d TGTGAGAGCCTAGAGATCCATTG
ITS1PhpFAM1 FAM-TCATTGAT-TGATAAGATCTTTGGGCAATGG-3IABlkFQ
a With standard PCR. b With qPCR. c For use with Ppm1/Ppa2 and Ppm1/Pme2. d For use with Ppm1/Ppm2. e Combined with Ppa2 from [145] in first round of nested PCR.

11. Genus Phytophthora

Phytophthora sojae (Kaufm. and Gerd.) causes seed decay, root rot, damping off that may occur before or after emergence, stem rot, and sometimes foliar blight [148]. Soybean is the only major host of P. sojae [149]. Another Phytophthora species, P. sansomeana, has been isolated since 1981 from soybean in the USA and China [150][151][152][153][154].
The first target for molecular diagnosis was the ITS region. One PCR assay developed for P. sojae utilized primers PS1/PS2 [155]. The primers were also used in a SYBR-green based qPCR assay with a 10 pg limit of detection. Another group using the primers found problems with discrimination against other Phytophthora species from soybean [156]. Consequently, they developed their own PSOJF1/PSOJR1 primers, also targeting the ITS region [157]. Other researchers [158] using these primers reported a limit of detection of 10 fg in absolute quantifications. Other targets described for P. sojae detection are a Ras-related protein (Ypt1) coding gene [159] and an A3aPro transposon-like element [160].
A hierarchical approach to Phytophthora genus- and species-specific qPCR assays based on mitochondrial genes [161] provides new targets. Here, two loci were used, one for detecting all Phytophthora spp., the tRNA locus (trnM-trnP-trnM), and another, atp9, and the spacer between atp9 and nad9 (atp9-nad9) for genus- and species-specific detection. This approach was utilized to design specific probes for many Phytophthora spp. [162]. The system was also adapted for the isothermal technique recombinase polymerase amplification (RPA) [163]. Building on these approaches, a diagnostic assay for P. sojae and P. sansomeana was developed [164]. Using a genus specific probe and probes specific to P. sojae and P. sansomeana this multiplex qPCR assay can simultaneously determine if a sample is infected by any Phytophthora spp. and if it contains either P. sojae, P. sansomeana, or both [164]. The assay is highly specific and sensitive. A plant mitochondrial internal control for quantification relative to soybean and to determine the presence of PCR inhibitors can also be included in the assay. Another artificial internal control can be added when testing soil samples. Primer sets for RPA also exist [164].
Other groups [165][166] used the Ty3/Gypsy retroelement as target. This transposable element is widely distributed in the Phytophthora genus and forms lineages that predate the separation of the species [167]. This sequence is a good target because it is present in all isolates and has multiple copies per genome. Primers PS12 and PS6R were developed for this sequence and produced a 282 bp amplicon in all P. sojae isolates, but not on other Phytophthora spp. and other fungal soybean pathogens, as well as soybean itself [165]. This was developed into a probe-based qPCR assay for P. sojae [166]. For additional information on the mentioned primers and assays, see Table 11.
Table 11. Primers and corresponding assays for diagnosis of Phytophthora spp. on soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS P. sojae a PS1 CTGGATCATGAGCCCACT 66 330 PCR/qPCR b [155]
PS2 GCAGCCCGAAGGCCAC
ITS P. sojae PSOJF1 GCCTGCTCTGTGTGGCTGT 50 127 qPCR b [157]
PSOJR1 GGTTTAAAAAGTGGGCTCATGATC
Ypt1 P. sojae F3 CCTTGTCTGCCCTCTCGA 65 b   LAMP [159]
B3 AGAAGCGTACACCCACCA
FIP GAATTTTCTGGGCGGGACAACGCCAGGATGGCTAAGGTTTCC
BIP GAGCTGGACGGCAAGACCATCCATAAGTGCGCTTAACCGG
LF GCACAATATTGTCAGCAACTGGATC
LB CAAGCTCCAGATTGTACGTTCA
A3aPro P. sojae F3 GCGTATTGAGGGTTGCTG 64 c   LAMP [160]
B3 GCGTCCTATCACCTAGTGC
FIP ACGTGGGTTCGGATTGGACC-CTTGGGTACTGTGTACCAG
BIP CGCCACCGATGATTCGACGA-AATCAACCATCACTCACCG
LB GTAGGATGATTGGATGAACAC
atp9 Phytophthora PhyG_ATP9_2FTail AATAAATCATAACCTTCTTTACAACAAGAATTAATG 57   multiplex qPCR [161][164]
nad9 PhyG-R6_Tail AATAAATCATAAATACATAATTCATTTTTATA
atp9-nad9 Phytophthora genus-specific TaqMan probe FAM-AAAGCCATC [ZEN] ATTAAACARAATAAAGC-IABkFQ
atp9-nad9 P. sojae P. sojae species-specific TaqMan probe HEX-TTGATATAT [ZEN] GAATACAAAGATAGATTTAAGTAAAT-IABkFQ
atp9-nad9 P. sansomeana P. sansomeana species-specific TaqMan probe Quasar670-TATTAGTACTAAYTACTAATATGCATTATTTTTAG-BHQ-2
tRNA-M Phytophthora TrnM-F ATGTAGTTTAATGGTAGAGCGTGGGAATC 39 d   RPA [163][164]
TrnM-R GAACCTACATCTTCAGATTATGAGCCTGATAAG
TrnM-P TAGAGCGTGGGAATCATAATCCTAATGTTG [FAM-dT] A [THF] G [BHQ1-dT] TCAAATCCTACCATCAT [3′-C3SPACER]
atp9   Atp9-F CCTTCTTTACAACAAGAATTAATGAGAACCGCTAT
atp9-nad9 P. sojae Psojae-nad9-R TTAAATCTATCTTTGTATTCATATATCAA
P. sansomeana Psan-nad9-R TTAGTAGTTAGTACTAATATAACAAAAATATAATA
atp9   Atp9-P TTGCTTTATTYTGTTTAATGATGGCWTTY (T-FAM) T [THF] A (T-BHQ1) YTTATTTGCTTTTT [3′-C3SPACER]
Ty3/Gypsy retroelement C. truncatum Pso12-F CAGGTTTTCAGCGATCTCATCCAAGTG 60 282 qPCR [166]
Pso6-R CACATTGCGGAAAAGGAGGTGATTGCT
Pso-P5 FAM-TGCCGACTGCGAGGTCAGCAACCACTTCAA-IBFQ
a Specificity contradicted by [156][157]. b Incubation for the LAMP assay. Incubation for 60 min. c Incubation for the LAMP assay. Incubation for 80 min. d Incubation for the RPA assay. Incubation for 29 min; details see [164].

12. Genus Pythium

Of the other Oomycete genus, Pythium, up to 14 species have been reported to infect soybeans [168]. Importantly, Pythium irregulare, P. sylvaticum, P. ultimum, and P. torulosum cause seed rot, seedling damping-off, and root rot [169]. Species-specific primers for detection of Pythium spp. by PCR were developed [170][171][172]. For detection of P. ultimum, a LAMP assay is also available [173]. For the primers and assays, see Table 12.
Table 12. Primers and corresponding assays for diagnosis of Pythium spp. on soybean.
Target Gene Target Species (Specificity) Primer/Probe (Combination) Sequence (5′-3′) Tm (°C) Fragment Length (bp) Assay Ref.
ITS P. ultimum and HS group K1 ACGAAGGTTGGTCTGTTG 55   PCR [170]
K3 TCTCTACGCAACTAAATGC
ITS P. aphanidermatum Pa1 TCCACGTGAACCGTTGAAATC 67

72		 210

150		 PCR [171]
none ITS2 GCTGCGTTCTTCATCGATGC
P. irregulare Pir1 AGCGGCGGGTGCTGTTGCAG
ITS P. aphanidermatum AsAPH2B GCGCGTTGTTCACAATAAATTGC 57 a 163

150		 PCR [172]
Pythium AsPyF CTGTTCTTTCCTTGAGGTG 52 a
P. torulosum AsTOR6 CGCCTGCCGAAACAGACTAG 59 a
Gene encoding a spore cell wall protein P. ultimum F3 CAACTGGAAAAGCAAGCGG 64 b   LAMP [173]
B3 CCGAAGAACTGTGTCCGC
FIP GAGCCAGACGGGCCAGTATCAAGTTACAGTGGCGTTGTCA
BIP TCTCTGTTGCTCGACTGGAGGGTTCCACCTCCTGTAAGACCT
F-Loop GCTTGCTCCAGTACGAATGC

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Subjects: Mycology
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :
Behnoush Hosseini
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Ralf Thomas Voegele
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Tobias Immanuel Link
View Times: 246
Revisions: 4 times (View History)
Update Date: 20 Jul 2023
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