Fungicide resistance
To assess boscalid resistance in Alternaria species commonly found in the primary potato and tomato cultivation regions of Russia, we carried out an extensive screening of isolates collected over a six-year period, from 2016 to 2021. The study encompassed a total of 42 isolates, distributed among various Alternaria species as follows: 16 isolates of A. linariae, 5 of A. solani, 9 of A. grandis, 10 of A. protenta, and 2 of A. alternariacida. For most of these isolates, the EC50 values were beyond the detectable range of the standard plate dilution series, indicating considerable variation in susceptibility (Table 3).
Most of Alternaria isolates were resistant to boscalid at all sampling sites and in all years, indicating that fungicide resistance is widespread. Levels of resistance differed considerably among strains of the same species and locality. Taken together, these results implicate that only several isolates of A. solani demonstrated sensitivity with EC50 values less than 5 µg/mL. Maximum EC50 value of A. solani isolates was 6,7 µg/mL (Table 3). Highly resistant strains with an EC50 exceeding 100 µg/mL were identified within all other tested groups. Isolates of A. linariae showed higher boscalid sensitivity, with mean EC50 of 182,5 µg/mL. A. protenta and A. alternariacida were also resistant, their average EC50 were 118,1 and 132,0 µg/mL, correspondingly. A moderate level of resistance to the fungicide was found in A. grandis (average EC50 −= 76,8 µg/mL) (Table 3).
While the EC50 values offer valuable insights into the fungicide sensitivity or resistance of these fungi, providing essential information for their management and control, we opted to present the colony diameters in Table 3. These measurements offer a concise overview of the overall resistance, providing additional context for interpreting our findings.
Table 3
The EC50 values, mean values, and standard deviation of Alternaria spp isolates cultivated on agar media supplemented with boscalid.
Species | | Colony growth diameter on media with the addition of boscalid, mm | EC50, µg/mL |
Strain | control | 1 µg/mL | 10 µg/mL | 100 µg/mL |
Alternaria grandis | A16UsPL21 | 59 | 49 | 45 | 30 | 103.0 |
A16PrPL22 | 62 | 49 | 40 | 20 | 60.5 |
A17VlPL41a | 59 | 52 | 43,6 | 21,7 | 68.0 |
A16KhPL41 | 61 | 50 | 44,5 | 25,3 | 75.6 |
mean values | | 60,25 ± 1,5 | 50 ± 1,4 | 43 ± 2,25 | 24,25 ± 4,4 | 76.8 |
Alternaria solani | A17SpbPL10 | 66,5 | 36 | 20 | 5,5 | 2.6 |
A21KrTL8 | 61,8 | 30,5 | 20,2 | 6,2 | 1.0 |
A21MTSt3 | 61 | 41,5 | 23,6 | 9,9 | 6.5 |
A17SpbPL11 | 60,7 | 39,9 | 24,8 | 9,3 | 6.7 |
A17SpbPL12 | 60,9 | 37,8 | 24 | 14,5 | 5.8 |
mean values | | 62,18 ± 2,45 | 37,14 ± 4,2 | 22,52 ± 2,2 | 9,08 ± 3,3 | 4.5 |
Alternaria alternariacida | A16PrPL21 | 61,5 | 40 | 40 | 36 | 133.8 |
A16PrPL22 | 60 | 45 | 40 | 35 | 130,09 |
mean values | | 60,75 ± 1 | 42,5 ± 3,5 | 40 | 35,5 ± 0,7 | 132,0 |
Alternaria protenta | A16PrPL11 | 44,5 | 45 | 45 | 20 | 90.0 |
A17VlPL41 | 44,3 | 44,1 | 44,3 | 16,1 | 79.0 |
A17KhPL51 | 44,3 | 44,1 | 44,1 | 17,6 | 83.9 |
A16PrPL45 | 44,3 | 44,1 | 44 | 17,9 | 85.3 |
A17VlPL31 | 44,3 | 44,2 | 44,4 | 25 | 117.1 |
A16UsPL31 | 44,3 | 44 | 44,3 | 34 | 210.5 |
A17VlPL51 | 44,2 | 44,3 | 44,2 | 27,2 | 127.0 |
A16KhPL11 | 44 | 44,4 | 44,3 | 26,2 | 135.0 |
A17VlPL51a | 44,3 | 44 | 44,1 | 29,1 | 157.4 |
mean values | | 44,27 ± 0,1 | 44,24 ± 0,3 | 44,3 ± 0,3 | 18,04 ± 1,44 | 118.1 |
Alternaria linariae | 7АHTF 11а | 60 | 57,6 | 55 | 40 | 160.0 |
А17АHTL 14e/2 | 68 | 55 | 50 | 41 | 170.0 |
A18MYKTL7 | 58,5 | 55,8 | 50,9 | 41 | 206.8 |
A18MYKTL18/1 | 57,2 | 54,4 | 53 | 40,8 | 207.6 |
А17АHTL3а* | 64,1 | 57,2 | 52,5 | 40,5 | 163.3 |
A17VlPL31a | 64,4 | 56,6 | 50,3 | 40,3 | 172.9 |
A17MYKTL10/1 | 55,3 | 55,2 | 52,7 | 40,9 | 201.0 |
A18MYKTL25/2(1) | 55,7 | 55 | 50,8 | 40,5 | 210.5 |
A17MYKTL11/2 | 54,8 | 57,1 | 50,2 | 40,2 | 215.2 |
A18BlTF1 | 62,8 | 57,1 | 52,5 | 40,3 | 165.7 |
A18VTL10/2 | 61,4 | 56,1 | 53 | 40,3 | 168.0 |
A16KhTL21 | 55,5 | 57,8 | 51,8 | 40,8 | 206.8 |
А18АКTL117/7 | 61,6 | 56,3 | 53,4 | 41 | 174.0 |
A20KrTL14 | 63,6 | 55,3 | 53 | 40,8 | 166.4 |
A20KrTL16 | 59,6 | 58,9 | 54,3 | 40,3 | 167.5 |
A21KrTS1.2 | 66,6 | 59,8 | 54,6 | 40,8 | 148.9 |
A21KrTL3 | 52 | 56,2 | 50,3 | 40,8 | 240.2 |
A21KrTL5 | 63,5 | 55,7 | 50,3 | 40,9 | 187.6 |
A21KrTL6 | 56,2 | 56,2 | 54,5 | 40,9 | 184.7 |
A21KrTL10 | 55,8 | 55,6 | 52 | 40,5 | 198.6 |
A21KrTS22 | 62,2 | 55,7 | 52,5 | 40,8 | 174.6 |
A21MTSt2 | 59,3 | 59,4 | 53,2 | 40,5 | 176.9 |
A21MTSt6 | 59,8 | 56,3 | 53,9 | 40,2 | 167.7 |
A21MTSt7 | 56,5 | 59,1 | 54,4 | 40,2 | 175.7 |
А21АHTL5 | 61,5 | 56,3 | 53,4 | 40,9 | 173.1 |
А21АHTL7 | 64,8 | 55,4 | 52,2 | 40,5 | 162.3 |
mean values | | 59,68 ± 4,3 | 56,91 ± 1,5 | 52,56 ± 1,6 | 40,60 ± 0,3 | 182.5 |
Sequence variation in the partial Sdh genes of Alternaria species
We sequenced 33 isolates of Alternaria species collected from both potato and tomato plants between 2016 and 2018. We obtained the complete sequence of the SdhB gene, which consists of 1082 base pairs. This gene contains three introns and an open reading frame (ORF) that encodes a protein comprising 308 amino acids. Within this gene, the analyzed sequences did not contain any known mutations associated with resistance (H278Y, H278R). However, several substitutions were identified within the gene, with some of them being common among all the isolates obtained from tomato plants. The nucleotide substitution at position 95 occurs within non-coding regions, introns, and does not result in amino acid alterations. The other substitutions identified do not cause changes in amino acid sequence. Therefore, no SNP results in a change in amino acid composition.
Upon sequencing a portion of the SdhC gene, we obtained sequences of 570 base pairs. These sequences consist of a single intron and an ORF that encodes a protein containing 160 amino acids. We discovered two substitutions in strains isolated from both potato and tomato plants. The substitution of G to A at nucleotide 123 is located within an intron and does not impact protein translation. Similarly, the C→T substitution at position 518 does not lead to changes in the protein sequence. Notably, no instances of the H134R substitution were found in the strains we examined.
The complete sequence of the SdhD gene spans 607 base pairs, comprising a single intron and an ORF encoding a 185-amino acid protein. Despite the absence of the H133R substitution associated with a higher boscalid resistance, the gene exhibits a series of other substitutions. Specifically, the SDHD gene coding region harbors eight unique SNPs (Fig. 3, Table 2). Noteworthy, these substitutions were consistently exclusive to all 16 strains isolated from tomato hosts.
A non-synonymous single nucleotide polymorphism (SNP) at codon 48 resulted in a dichotomous encoding of amino acids: valine for potato-derived isolates and isoleucine for A. linariaea originating from tomato (Fig. 5, Table 4).
Consequently, our findings reveal that three SNPs within SdhB and eight within SdhD effectively discriminate between isolates collected from potato and tomato hosts (Table 6). Sequence scrutiny of the Sdh gene subunits unveils a high degree of phylogenetic relatedness among these isolates compared to their correlations with other Alternaria species (A. alternata and A. brassicae).
Table 6
Single nucleotide polymorphisms (SNPs) detected in coding and non-coding regions of sdh genes, distinguishing species and strains derived from tomato and potato hosts.
Amino acid | Amino acid position | Polymorphism | Host | Alternaria species | Location |
Threonine (T) | 28 | ACT | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
ACC | S. lycopersicum | A. linariaea |
Serine (S) | 46 | TCA | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
TCC | S. lycopersicum | A. linariaea |
Valine (V) | 48 | GTC | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
Isoleucine (I) | ATC | S. lycopersicum | A. linariaea |
Proline (P) | 67 | CCA | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
CCC | S. lycopersicum | A. linariaea |
Proline (P) | 80 | CCC | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
CCA | S. lycopersicum | A. linariaea |
Isoleucine (I) | 106 | ATT | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
ATC | S. lycopersicum | A. linariaea |
Leucine (L) | 126 | CTA | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
CTC | S. lycopersicum | A. linariaea |
Leucine (L) | 130 | CTC | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | sdhD exon |
CTG | S.lycopersicum | A. linariaea |
Proline (P) | 176 | CCG | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | SdhB exon |
CCA | S. lycopersicum | A. linariaea |
Aspartic acid (D) | 257 | CGA | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | SdhB exon |
CGC | S. lycopersicum | A. linariaea |
Asparagine (N) | 270 | AAC | S. tuberosum | A. alternar., A. gr., A. prot., A. sol. | SdhB exon |
AAT | S. lycopersicum | A. linariaea |