Phytotoxins Produced by Two Biscogniauxia rosacearum Strains, Causal Agents of Grapevine Trunk Diseases, and Charcoal Canker of Oak Trees in Iran

Biscogniauxia rosacearum, recognized for the first time as a pathogen involved in grapevine trunk diseases in Paveh (west of Iran) vineyards, produced meso-2,3-butanediol (1) as the only phytotoxin. Nectriapyrone (2), (3R)-5-methylmellein (3), (3R)-5-methyl-6-methoxymellein (4), and tyrosol (5) were instead produced as phytotoxins from a strain of the same fungus isolated from oak trees in Zagros forests of Gilan-e Gharb, Kermanshah Province. They were identified comparing their 1H and 13C NMR, ESIMS, and specific optical rotation data with those already reported in the literature. The phytotoxicity of metabolites (1–5) was estimated by leaf puncture assay on Quercus ilex L. and Hedera helix L., and by leaf absorption assay on grapevine (Vitis vinifera L.) at a concentration of 5 × 10−3 and 10−3 M. Tested on grapevine, meso-2,3-butanediol (1) and (3R)-5-methyl-6-methoxymellein (4) resulted to be the most phytotoxic compounds. On Q. ilex, nectriapyrone (2) and tyrosol (5) showed severe necrosis at the highest concentration while none of the compounds (1–5) was active on H. helix. Furthermore, the phytotoxicity of compounds 3 and 4 was also compared with that of some related natural melleins to perform a structure-activity relationship (SAR) study. The results of this study were also discussed.

Recently, massarilactones D and H were isolated for first time as phytotoxins produced by Kalmusia variispora, responsible for GTDs in Iran [11]. The symptoms of the same disease in Iran were also induced by Didymella glomerata and Truncatella angustata, which

Results and Discussion
B. rosacearum strains IRAN 4194C and IRAN 4287C were grown in vitro, and their culture filtrates were extracted with EtOAc. The preliminary investigation of their organic extracts revealed a substantial difference in the toxins production. Thus, these organic extracts were fractionated, (see Material and Methods Section) to give five homo-Toxins 2021, 13, 812 3 of 12 geneous metabolites. The grapevine strain of B. rosacearum IRAN 4194C produced only one main metabolite, while the other four compounds were isolated from the oak strain B. rosacearum IRAN 4287C. They were identified as meso-2,3-butanediol, nectriapyrone, (3R)-5-methylmellein, (3R)-5-methy-6-methoxymellin, and tyrosol (1-5, Figure 1) by comparison of their 1 H and 13 C NMR and ESI MS spectra, and were needed the specific optical rotations with the data already reported in literature (see below).
Toxins 2021, 13, x FOR PEER REVIEW metabolites. The grapevine strain of B. rosacearum IRAN 4194C produced only one metabolite, while the other four compounds were isolated from the oak strain B. ros rum IRAN 4287C. They were identified as meso-2,3-butanediol, nectriapyrone, (3 methylmellein, (3R)-5-methy-6-methoxymellin, and tyrosol (1-5, Figure 1) by compa of their 1 H and 13 C NMR and ESI MS spectra, and were needed the specific optical tions with the data already reported in literature (see below). In particular, the 1 H NMR of 1, a symmetric compound, showed the quartet (J Hz) at δ 3.80, typical of the protons (H-2 and H-3) bonded to a secondary hydroxy carbon, which coupled with the protons of the adjacent methyl group (H3-C1 and H resonating as a doublet (J = 6.5 Hz) at δ 1.14 [24]. The 13 C NMR spectrum showe signals of the oxygenated carbons (C-2 and C3) at the typical values of δ 70.8 and tho the two methyl carbons (C-1 and C-4) at δ 16.8 [25]. The ESI MS spectrum showe significant ions generated from the protonated molecular adduct ion by loss of wate +H -H2O] + at m/z 73. Furthermore, compound 1 did not show optical activity, thus i identified as the meso-butanediol (1), whose spectroscopic data were in agreement those reported by Gallwey at al. 1990 [26]. In particular, the 1 H NMR of 1, a symmetric compound, showed the quartet (J = 6.5 Hz) at δ 3.80, typical of the protons (H-2 and H-3) bonded to a secondary hydroxylated carbon, which coupled with the protons of the adjacent methyl group (H 3 -C1 and H 3 -C4) resonating as a doublet (J = 6.5 Hz) at δ 1.14 [24]. The 13 C NMR spectrum showed the signals of the oxygenated carbons (C-2 and C3) at the typical values of δ 70.8 and those of the two methyl carbons (C-1 and C-4) at δ 16.8 [25]. The ESI MS spectrum showed the significant ions generated from the protonated molecular adduct ion by loss of water [M +H -H 2 O] + at m/z 73. Furthermore, compound 1 did not show optical activity, thus it was identified as the meso-butanediol (1), whose spectroscopic data were in agreement with those reported by Gallwey et al., 1990 [26].
(3R)-5-Methylmellin was characterized by 1 H and 13 C NMR and ESI MS spectra, but also by measuring its specific optical rotation, which is in agreement with the value previously reported by Okuno et al., 1986 [35]. In particular, its 1 H NMR spectrum showed a singlet at δ 10.98 due to a hydroxyl group at C-8 hydrogen bonded with the C-1 carbonyl group and two doublets (J = 8.4 Hz) at δ 7.28 and 6.82, which are typical signals of two ortho-coupled aromatic protons (H-6 and H-7, respectively) of a tetrasubstituted benzene ring with H-7 hupfield shifted for the electronic effect of the ortho-located HO-C8. In addition, a multiplet of a proton (H-3) of a secondary oxygenated carbon appeared at δ 4.69, which coupled with the protons of the adjacent methylene group (H 2 C-4), resonating as two double doublets (J = 16.5 and 2.0 Hz and J =16.5 and 11.9 Hz) at δ 2.95 and 2.72. H-3 also coupled with the protons of the geminal methyl group (Me-C3), appearing as a doublet (J = 7.2 Hz) at δ 1.84 [24]. The 13 C NMR spectrum showed the singlet of the ester carbonyl group (O = C-1) at δ 170.1 together with the signals of the protonated secondary carbons, two of which are aromatic (C-6 and C-7) and the other one aliphatic (C-3) at δ 137.4, 115.2, and 74.8. C-7) was up-field shifted for the electronic effect of the HOC-8,. The carbons of the methylene group (C-4), those of the vinylic methyl group (Me-C5) and of the aliphatic one (MeC-3) were observed at δ 31,5, 16.2 and 20,6, respectively. The aromatic tertiary sp 2 carbons, one which was oxygenated, resonated at δ 160.0, 137.6 137.3, and 108.3 for C-8, C-5, C-4a, and C-8a, with the last up-field shifted as reported for C-7 [25]. Its ESI MS spectrum showed the protonated adduct ion [M + H] + at m/z 193. These data were in agreement with those reported by Okuno et al., 1986 [35].
(3R)-5-methyl-6-methoxymellein (4) showed a specific optical rotation in agreement to the value reported by de Alvarenga et al., 1978 [36]. It was identified by 1 H and 13 C NMR and ESI MS data. In particular, its 1 H NMR spectrum showed the singlet of the HO-C 8 at δ 11.36 being hydrogen bonded, as in 3, with the carbonyl group at C-1 and another singlet typical of a proton (H-7) of a pentasubstituted benzene ring at δ 6.38. The latter signal was upfield shifted for the electronic effect of the ortho-hydroxyl group at C-8. The same spectrum showed the other two singlets at δ 3.85 and 2.03, typical of a methoxy group and a vinyl methyl (MeC-5) together with the multiplet of the proton (H-3) of an oxygenated secondary carbon resonating as a multiplet at δ 4.61. H-3 coupled with the protons of the adjacent methylene group (H 2 C-4) and those of germinal methyl group (Me-C3), which were observed as two double doublets (J = 16.7 and 2.0 Hz, and J = 16.7 and 11.9 Hz) and as a doublet (J = 6.3 Hz) at δ 2.97, 2.68 and 1.56, respectively [24]. The 13 C NMR spectrum showed the singlet of the ester carbonyl group (C-1) and the signals of secondary carbons (C-7 and C-3) and of the methylene group (H 2 C-4) at δ 115.1, 74.8, and 31.9. The signals of the methoxy group (OMe), of the vinyl methyl (Me-C5), and of the aliphatic one (Me-C3) were observed at δ 55.7, 10.6, and 20.9, respectively. The five aromatic tertiary sp 2 carbons, two of which were oxygenated, appeared at δ 164.6, 162.9, 137.6, 137.4, and 114.7 for C-6, C-8, C-5, C-4a, and C-8a. This latter signal appeared upfield shifted for the electronic effect of the ortho-located hydroxyl group at C-8, which similarly affects the chemical shift of C-7 [25]. The ESIMS spectrum showed the dimer sodiated [2M + Na] + and the protonated [M + H] + adduct ions at m/z 467 and 223. These data are in agreement to those previously reported by de Alvarenga et al., 1978 [36].
The ortho-location of the methoxy group and the vinyl methyl at C-6 and C-5, respectively, was determined by the correlations observed in the NOESY spectrum [37] between the methoy group with H-7 and that of Me-C5 with H 2 -C-4. The correlation between the methoxy group with MeC-5 was probably not observed as the first is oriented toward H-7 and its rotation is almost hindered.
(3R)-5-methyl and (3R)-5-methyl-6-methoxy-mellein (3 and 4) both belong to the group of 4-dihydroisocoumarins and are very well known as naturally occurring compounds. They are important metabolites for the producer organisms and are involved in many biological activities including phytotoxicity [38].
Tyrosol (5) was identified by comparing its 1 H and 13 C NMR and ESIMS data with those previously reported Reveglia et al., 2021 [9]. In particular, its 1 H NMR spectrum showed a couple of doublets (J = 8.0 Hz) of the two aromatic ortho-coupled protons of a p-disubstituted benzene ring at δ 7.20 (H-2 and H-6) and 6.80 (H-3 and H-5) with the last signal upfield shifted for the electronic effect of the hydroxyl group ortho-located at C-4. The same spectrum showed the two triplets (J = 6.4 Hz) of the two methyelene groups of 2-hydroxy ethyl side chain observed at δ 3.80 (H 2 C-2') and 2.80 (H 2 C-1') [24]. The 13 C NMR spectrum showed the overlapped signals of the protonated aromatic carbons (C2/C6) and (C3/C5) at δ 113.0 and 118.5, with the latter upfield shifted for the electronic effect already described by the hydroxyl group at C-4, and the two methylene carbons of the side chain at δ 65.4 and 39.6 for C-2'and C-1', respectively. The two tertiary sp 2 aromatic carbons, one of which is oxygenated, resonated at δ 156.5 and 133.6, for C-4 and C-1, respectively [25]. It ESIMS spectrum exhibited the dimer sodiated [2M + Na] + and the sodiated [M + Na] + adduct ions at m/z 299 and 139, respectively. These data were in agreement to those previously reported [9].
The phytotoxic activity of compounds (1)(2)(3)(4)(5) were estimated by leaf puncture assay on Quercus ilex L. and Hedera helix L., and by leaf absorption assay on grapevine (Vitis vinifera L.) at a concentration of 5 × 10 −3 M and 10 −3 M. The results of these assays are reported in Table 1. In the leaf absorption assay, meso-2,3-butanediol (1) and (3R)-5methyl-6-methoxymellein (4) resulted to be the most phytotoxic compounds (Figure 2). In the leaf puncture assays, nectriapyrone (2) and tyrosol (5) induced severe necrosis at the highest concentration while none of the compounds (1-5) were active on H. helix L (Figure 3).       Furthermore, the activity of compounds 3 and 4 was also compared with that of some related melleins, namely (3R)-mellein (6) and (3R,4R)-and (3R,4S)-4-hydroxy melleins (7 and 8), isolated fom Sardiniella urbana, [45] and (3R)-6-hydroxymellein (9), isolated from Phoma chenopodiicola, as previously described [46]. Also, the (3R)-6-methoxymellein (10), which was prepared by methylation starting from 9 by reaction with an ether solution of diazomethane, was used. The results of this structure-activity relationship (SAR) study, reported in Table 1, showed that in the toxicity on grapevine, the hydroxy group at C-4 of pyranone moiety negatively affected the phytotoxicity on V. vinifera L. Instead, the C-6 substitution of the aromatic ring either with a phenolic hydroxy or a methoxy group is an important feature to cause phytotoxicity on the same plant. The results obtained on Quercus ilex L. (Table 1) suggested that the absence of any substituents on the aromatic ring is essential feature for the toxicity, demonstrating a different mode of action of the melleins on grapevine and oak leaves. (1) is the only phytotoxin synthesized by Biscogniauxia rosacearum (IRAN 4194C), which was recognized for the first time as a pathogen involved in GTDs in Paveh, Kermanshah Province (west of Iran) vineyards. Similarly, nectriapyrone (2), (3R)-5-methylmellein (3), (3R)-5-methyl-6-methoxymellein (4), and tyrosol (5) were instead produced as phytotoxins from a strain of the same fungus (IRAN 4287C) isolated from oak trees in Zagros forests of Gilan-e Gharb, Kermanshah Province. Tested on grapevine (Vitis vinifera L.), meso-2,3-butanediol and (3R)-5-methyl-6-methoxymellein resulted to be the most phytotoxic compounds, while nectriapyrone (2) and tyrosol (5) showed severe necrosis at the highest concentration when assayed on oak (Quercus ilex L.) leaves. On ivy (H. helix L), none of the compounds (1-5) were active. The results of SAR study using melleins 3,4 and 6-10 showed that the hydroxy group at C-4 of pyranone ring negatively affected the phytotoxicity on V. vinifera L., while the C-6 substitution of benzene ring either with a phenolic hydroxy group or a methoxy group is determinant for the phytotoxicity. On Quercus ilex L., the absence of any substituents on the aromatic ring is an essential feature to impart phytotoxic activity. These results suggested a different mode Furthermore, the activity of compounds 3 and 4 was also compared with that of some related melleins, namely (3R)-mellein (6) and (3R,4R)-and (3R,4S)-4-hydroxy melleins (7 and 8), isolated fom Sardiniella urbana, [45] and (3R)-6-hydroxymellein (9), isolated from Phoma chenopodiicola, as previously described [46]. Also, the (3R)-6-methoxymellein (10), which was prepared by methylation starting from 9 by reaction with an ether solution of diazomethane, was used. The results of this structure-activity relationship (SAR) study, reported in Table 1, showed that in the toxicity on grapevine, the hydroxy group at C-4 of pyranone moiety negatively affected the phytotoxicity on V. vinifera L. Instead, the C-6 substitution of the aromatic ring either with a phenolic hydroxy or a methoxy group is an important feature to cause phytotoxicity on the same plant. The results obtained on Quercus ilex L. (Table 1) suggested that the absence of any substituents on the aromatic ring is essential feature for the toxicity, demonstrating a different mode of action of the melleins on grapevine and oak leaves. (1) is the only phytotoxin synthesized by Biscogniauxia rosacearum (IRAN 4194C), which was recognized for the first time as a pathogen involved in GTDs in Paveh, Kermanshah Province (west of Iran) vineyards. Similarly, nectriapyrone (2), (3R)-5-methylmellein (3), (3R)-5-methyl-6-methoxymellein (4), and tyrosol (5) were instead produced as phytotoxins from a strain of the same fungus (IRAN 4287C) isolated from oak trees in Zagros forests of Gilan-e Gharb, Kermanshah Province. Tested on grapevine (Vitis vinifera L.), meso-2,3-butanediol and (3R)-5-methyl-6-methoxymellein resulted to be the most phytotoxic compounds, while nectriapyrone (2) and tyrosol (5) showed severe necrosis at the highest concentration when assayed on oak (Quercus ilex L.) leaves. On ivy (H. helix L), none of the compounds (1-5) were active. The results of SAR study using melleins 3,4 and 6-10 showed that the hydroxy group at C-4 of pyranone ring negatively affected the phytotoxicity on V. vinifera L., while the C-6 substitution of benzene ring either with a phenolic hydroxy group or a methoxy group is determinant for the phytotoxicity. On Quercus ilex L., the absence of any substituents on the aromatic ring is an essential feature to impart phytotoxic activity. These results suggested a different mode of action of the melleins on grapevine and oak leaves. Further studies need to be performed on the phytotoxins/plant-pathogen interaction.

Fungal Strains
B. rosacearum strain IRAN 4194C was obtained from vineyards of Paveh, Kermanshah Province, showing grapevines trunk diseases symptoms while the strain IRAN 4287C was obtained from oak trees showing charcoal canker and decline in Kermanshah Province (Gilan-e Gharb, Iran), respectively. DNA extraction, PCR, and maximum parsimony analysis were performed as previously reported [23]. ITS region of ribosomal DNA and a part of β-tubulin gene (tub2) were amplified for identification of the isolates. Sequences of both strains IRAN 4194C (ITS: MW786620) and IRAN 4287C (ITS: MZ359663; tub2: MZ362432) were deposited in GenBank. Their pathogenicity Koch's postulates were followed under greenhouse conditions (22-28 • C). Fungal strains were deposited in collection of the Iranian Research Institute of Plant Protection (Tehrean, Iran) (IRAN).

Leaf Puncture Assay
Compounds 1-10 were tested on Quercus ilex L. and Hedera helix L., using the leaf puncture assay at concentrations of 5 × 10 −3 and 10 −3 M. The compounds were dissolved in MeOH and then the solution was diluted with MilliQ H 2 O to reach the required concentration with a final concentration of MeOH at 4%. On the adaxial surface of the plant leaves, which were previously punctured with a sterile needle, a droplet (20 µL) of compound solutions was applied. The leaves were placed on the surface of a water-saturated filter paper in Petri dishes. The negative control was a solution of 4% MeOH in MilliQ H 2 O . The dishes were sealed with parafilm and incubated at 24 • C for 7 days in a temperature-regulated chamber. For each metabolite and plant species tested, three replications were performed. Seven days after treatment, necrotic lesion development was evaluated using a visual 0-3 scale (0 = no necrosis; 1 = slight necrosis; 2 = intermediate necrosis; 3 = severe necrosis).

Leaf Absorption Assay
Grapevine leaves (Vitis vinifera L.) were used for this assay. Briefly, the cuttings were placed in a tube containing the solutions of compounds 1-10 in 4% of MeOH/MilliQ H 2 O and tested at two concentrations (5 × 10 −3 M and 10 −3 M). Twelve hours after, the leaves were moved to another tube containing only MilliQ-H 2 O. The symptoms were evaluated after 48 h using a 0-3 scale (0 = no symptoms; 1 = slight symptoms; 2 = intermediate symptoms; 3 = complete wilting). A solution of 4% MeOH in MilliQ H 2 O was used as negative control. The experiment was carried out in triplicate.