Biosci. Biotechnol. Biochem., 68 (5), 1125–1130, 2004

Novel Fusicoccins R and S, and the Fusicoccin S Aglycon (Phomopsiol) from Phomopsis amygdali Niigata 2-A, and Their Seed Germination-stimulating Activity in the Presence of Abscisic Acid

y Naoto TAJIMA,1 Manabu NUKINA,1;2 Nobuo KATO,3 and Takeshi SASSA1;2;

1Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University), Ueda-cho, Morioka 020-8550, Japan 2Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan 3Institute of Scientific and Industrial Research, Osaka University, Mihogaoka-cho, Ibaraki 567-0047, Japan

Received January 13, 2004; Accepted February 2, 2004

Our search for new 3-hydroxyfusicoccins structurally Fusicoccin A (FC A, Fig. 1), a unique metabolite from related to cotylenin A from a culture of Phomopsis Phomopsis (Fusicoccum) amygdali,1,2) is a novel 5-8-5- amygdali Niigata 2-A resulted in the isolation of novel 3- membered tricyclic diterpene glucoside3,4) possessing hydroxy fusicoccins, called fusicoccins R and S, and the potent plant-growth stimulating acitivity.5) The mode of fusicoccin S aglycon, called phomopsiol, together with action of FC A on plant 14-3-3 protein and the Hþ- known 3 -hydroxyfusicoccin J. The structure of pho- ATPase are currently attracting much attention.6) We mopsiol was identified as that of O-demethyl-3-epicotyl- have reported in a previous paper the isolation of new enol based on spectroscopic evidence. The structures of polar fusicoccins P and Q, and 3-epifusicoccins H and Q fusicoccins R and S were also determined to be those of from the 30-deacetyl-FC A-producing fungus, Phomop- 30-deacetyl-3 -hydroxyfusicoccin A and 3 -hydroxy-3- sis amygdali Niigata 2-A (an isolate of a peach epifusicoccin H. The lettuce seed germination-stimulat- Fusicoccum canker fungus), together with the presence ing activity of fusicoccins R and S, phomopsiol and 3 - of two fusicoccin-biosynthetic pathways finally giving hydroxyfusicoccin J was examined in the presence of FC A and its 30-deacetyl derivative, as well as 16-O- ABA; fusicoccin R and 3 -hydroxyfusicoccin J were demethyl-3-epi-FC J.7) highly active, while fusicoccin S and phomopsiol were We have recently found that cotylenin A (CN A, inactive. The possible biosynthetic relationships among Fig. 1),8) the sole fusicoccin-congener isolated as a these novel fusicoccins having a 3 -or3 -hydroxy potent plant-growth regulator from Cladosporium sp. group in their diterpene moiety are briefly discussed. 507W,9,10) induced the differentiation of human acute myeloid leukemia cells11) and apoptosis in human lung Key words: fusicoccin R; fusicoccin S; phomopsiol; carcinoma cells in the presence of interferon-.12) These Phomopsis amygdali; seed germination useful biological activities of CN A toward mammalian

O OAc O OH 2' 4' 4'' O 3' O HO OH 5'' HO HO OH O O O 1' 5' 6' O OMe 20 1'' OH 15 HO O 19 2'' 3'' HO O HO O 17 14 8 9 OAc 7 10 13 H 6 11 H H 5 12 2 1 3 18 OH 4 OMe OMe OH 16 HO

Fusicoccin A Cotylenin A Fusicoccin H (FC A) (CN A) (FC H)

Fig. 1. Structures of Fusicoccins A and H, and Cotylenin A.

y To whom correspondence should be addressed. Fax: +81-235-28-2862; E-mail: [email protected] 1126 N. TAJIMA et al. cells prompted us to search for new CN-type fusicoccins plate. having the 3-hydroxy group from the Niigata 2-A Substance A was detected together with 16-O-de- fungus. Based on the isolation of 3-hydroxy-FC J from methyl-3-epi-FC J in fraction 3 (15:1 CHCl3/EtOH). an Italian fungus,13) we tried to generate CN-type Fraction 3 was rechromatographed by a similar method, fusicoccins by the fungus in a stationary culture using using a 19:1 mixture of CHCl3/EtOH and then a 31:1 rice bran and wheat bran, and succeeded in isolating mixture of EtOAc/EtOH, to give fine colorless crystals novel 3-hydroxyfusicoccins, called fusicoccin R (FC R) (18 mg). Substances B and C were detected together 14) and fusicoccin S (FC S), and the FC S aglycon, called with 16-O-demethyl-FC J in fraction 4 (7:1 CHCl3/ phomopsiol. This paper reports the isolation and EtOH). These were separated by a similar method, using structures of FC R, FC S and phomopsiol, together with EtOAc/EtOH mixtures, to give 5 fractions. Fraction 20 that of 3-hydroxy-FC J from the Niigata 2-A fungus (31:1 EtOAc/EtOH) was crystallized from EtOAc to (Fig. 3). FC R and 3-hydroxy-FC J having the 16-O- give substance B (3.3 mg) as fine colorless crystals. methyl group showed high germination-stimulating Fraction 40 (15:1 EtOAc/EtOH) was further purified by activity toward lettuce seeds in the presence of ABA; a similar method, using a 10:1 mixture of EtOAc/ FC S and phomopsiol having no 16-O-methyl group in acetone, to give substance C (13.9 mg) as a colorless the 3-epimeric structures were inactive in this assay. The solid. Substance D was detected together with FC Q in isolation of these 3-hydroxyfusicoccins and phomopsiol fraction 9 (3:1 CHCl3/MeOH). Fraction 9 was separated suggested the presence of new fusicoccin-biosynthetic by a similar method, using a 10:1 mixture of EtOAc/ pathways in the fungus; their possible biosynthetic EtOH, and was further purified by preparative silica gel relationships are discussed (Fig. 3). TLC, using a 2:1 mixture of EtOAc/n-BuOH, to give substance D (6.3 mg) as a colorless solid. In this Materials and Methods experiment, fraction 2 (ca. 2.8 g) containing a 1:1 mixture of the major metabolites of 30-deacety-FC A and General procedures. Optical rotation values were FC J was separated. measured with a Horiba SEPA-300 digital polarimeter. Substance A (Phomopsiol): Mp 120–121 C (EtOAc); 1 13 21 H- and C-NMR spectra were recorded with a Jeol ½D 28 (c 0.12, MeOH). FAB-MS m=z: 359 EX-400 spectrometer. The DIF-NOE spectra of FC S ½M þ Naþ. HR-FABMS m=z ½M þ Naþ: calcd. for 1 and phomopsiol were recorded with a Jeol Lambda-600 C20H32O4Na, 359.2198; found, 359.2201. The H- and spectrometer. FABMS and HR-FABMS spectra were 13C-NMR data are shown in Table 1. recorded with a JMS-700 mass spectrometer. Wako FC- Substance B (3-Hydroxy-FC J):13) Mp 134–135 C 25 40 silica gel was employed for flash chromatography, (EtOAc); ½D þ18 (c 0.20, CHCl3). FAB-MS m=z: þ 1 and silica gel TLC plates (Merck F254 Nos. 5554 and 619 ½M þ Na . H-NMR (400 MHz, CDCl3) : 0.81 (d, 5583) were respectively employed for the TLC analysis J ¼ 7:2 Hz, H17), 0.98 (d, 6.8, H20), 1.07 (d, 6.8, H19), and preparative TLC. 1.20 (s, H18), 1.27 (s, H500), 1.28 (s, H400), 2.95 (m, H6), 3.16 (d, 9.6, H16), 3.17 (sep, 6.8, H15), 3.40 (d, 9.6, Extraction and isolation of the new substances. P. H16), 3.41 (O-Me), 3.48 (t, 9.6, H30), 3.52 (dd, 9.6, 4.0, amygdali Niigata 2-A was cultured in the stationary H60), 3.62 (dd, 9.6, 4.0, H20), 3.79 (d, 11.2, H9), 3.83 (t, mode for 21 days at 25 C in 500-ml flasks as a merlstem 8.8, H40), 3.96 (dd, 11.2, 4.0, H8), 4.98 (d, 4.0, H10), culture, each containing rice bran (26 g), wheat bran 5.15 (br.d, 17.6, H300), 5.17 (br.d, 10.8, H300), 5.54 (d, (13 g) and deionized water (40 ml). The mycelia and 2.4, H1), 5.77 (dd, 17.6, 10.8, H200); 13C-NMR media obtained from 80 flasks were homogenized in (100 MHz, CDCl3) : 8.6 (C17), 20.6 (C20), 21.2 acetone. The aq. acetone solution (10 liters) obtained by (C19), 23.7 (C18), 25.3 (C500), 25.9 (C400), 28.0 (C15), filtration was concentrated in vacuo. The residual aq. 31.7 (C4), 35.3 (C13), 35.4 (C5), 41.0 (C6), 41.2 (C7), solution (700 ml) was adjusted to pH 9.0 with 5% aq. 53.5 (C11), 59.3 (O-Me), 64.1 (C60), 69.3 (C40), 71.9 0 0 0 00 Na2CO3 and extracted 3 times with EtOAc (1.4 liters (C5 ), 73.6 (C2 ), 73.8 (C3 ), 76.2 (C1 ), 77.3 (C16), each) after being saturated with NaCl. The EtOAc 77.8 (C8), 78.2 (C9), 81.7 (C12), 82.5 (C3), 101.3 (C10), extract (34.5 g) obtained by evaporating EtOAc was 114.8(C300), 128.2(C1), 134.5(C10), 142.7(C2), 144.1 washed 3 times with n-hexane (200 ml each) and then 3 (C200), 146.0 (C14). 25 times with a 2:1 mixture of n-hexane/ether (200 ml Substance C (FC R): ½D þ3:7 (c 0.050, CHCl3). each). The obtained residual extract (6.1 g) was sepa- FAB-MS m=z: 677 ½M þ Naþ. HR-FABMS m=z þ rated by silica gel flash chromatography with CHCl3/ ½M þ Na : calcd. for C34H54O12, 677.3514; found, 1 13 EtOH mixtures and then a 3:1 mixture of CHCl3/MeOH 677.3513. The H- and C-NMR data are shown in to give 10 fractions. The CN-type substances included in Table 1. 24 the fractions could be detected by TLC with a vanillin- Substance D (FC S): ½D þ27:8 (c 0.030, MeOH). þ conc. H2SO4 spray reagent; these CN-type substances FAB-MS m=z: 521 ½M þ Na . HR-FABMS m=z þ and related metabolites gave purple spots on a TLC plate ½M þ Na : calcd. for C26H42O9, 521.2727; found, immediately after spraying with the reagent, while 521.2723. The 1H- and 13C-NMR data are shown in fusicoccins produced no spots without heating the TLC Table 1. Novel Fusicoccins R and S, and Phomopsiol and Their Germination-stimulating Activity 1127 Table 1. 1H- and 13C-NMR Assignments of Fusicoccin R (FC R), Fusicoccin S (FC S) and Phomopsiol

FC Re FC S Phomopsiol Position a,c b,c a,d b,d a,d b,c C H C H C H 1 127.9 5.54 (d, 2.4) 134.5 5.32 (d, 2.2) 134.3 5.48 (d, 2.4) 2 144.7 141.0 141.0 3 82.4 83.7 83.8 4 31.7 1.97–2.07 (m) 35.3 1.57–1.66 (m) 35.4 1.79 (ddd, 12.8, 7.0, 7.0) 1.44–1.54 (m) 1.53 (ddd, 12.0, 7.0, 7.0) 1.66 (ddd, 12.8, 7.0, 7.0) 5 35.5 1.97–2.07 (m) 34.1 1.78 (m) 34.2 1.90 (m) 1.30–1.40 (m) 1.47 (m) 1.60 (m) 6 41.1 2.92 (m) 42.2 2.81 (m) 42.2 2.88 (m) 7 41.1 1.97–2.07 (m) 43.7 1.88 (m) 44.9 2.03–2.09 (m) 8 77.3 3.93 (dd, 10.0, 4.5) 79.7 3.91 (dd, 10.1, 4.4) 78.7 3.96 (dd, 10.0, 4.6) 9 78.0 3.88 (d, 10.0) 78.6 3.81 (d, 10.1) 68.2 4.05 (d, 10.0) 10 138.9 137.3 138.6 11 53.9 53.3 53.1 12 81.0 3.77–3.83 (m) 43.5 1.57–1.66 (m) 43.7 1.56–1.68 (m) 1.72 (m) 13 36.7 2.35 (dd, 15.5, 6.3) 27.6 2.04 (ddd, 15.7, 8.0, 3.2) 27.8 2.14 (15.5, 5.6, 5.6) 2.16 (dd, 15.5, 6.4) 1.98 (ddd, 15.7, 8.4, 7.4) 2.03–2.09 (m) 14 139.9 150.2 150.2 15 33.0 3.34 (m) 29.1 3.18 (sep, 6.8) 29.0 3.28 (sep, 6.8) 16 77.3 3.40 (d, 9.3) 68.8 3.33 (d, 11.6) 68.9 3.52 (d, 11.3) 3.19 (d, 9.3) 3.26 (d, 11.6) 3.40 (d, 11.3) 17 8.6 0.83 (3H, d, 7.3) 9.9 0.86 (3H, d, 7.1) 10.0 0.94 (3H, d, 7.3) 18 23.7 1.24 (3H, s) 27.0 1.18 (3H, s) 26.7 1.23 (3H, s) 19 68.1 4.31 (dd, 10.4, 4.2) 22.0 1.00 (3H, d, 6.8) 21.5 1.03 (3H, d, 6.8) 3.77–3.83 (m) 20 15.5 1.09 (3H, d, 6.8) 20.6 0.88 (3H, d, 6.8) 20.3 0.96 (3H, d, 6.8) 10 101.3 5.07 (d, 4.0) 103.6 4.84 (d, 3.8) 20 72.1 3.63 (dd, 9.6, 4.0) 73.7 3.34 (dd, 9.2, 3.8) 30 73.7 3.77–3.83 (m) 75.0 3.61 (dd, 9.2, 9.2) 40 72.3 3.38–3.43 (m) 71.1 3.32 (dd, 9.2, 9.2) 50 71.0 3.66 (dt, 9.8, 4.9) 73.7 3.54–3.58 (m) 60 63.1 3.49 (dd, 10.0, 4.9) 62.0 3.63 (dd, 11.9, 3.8) 3.47 (dd, 10.0, 4.9) 3.54–3.58 (m) 100 75.8 200 143.2 5.79 (dd, 17.6, 11.2) 300 114.4 5.14 (dd, 11.2, 0.7) 5.13 (dd, 17.6, 0.7) 400 25.8 1.27 (3H, s) 500 25.4 1.27 (3H, s)

a b c d e 100 MHz; 600 and 400 MHz (J in Hz); CDCl3 solution; CD3OD solution; methoxyl signal: C 59.30, H 3.40; acetyl signal: C 21.1 and 172.0, H 2.10.

Table 2. Germination-stimulating Activity of Fusicoccin J (FC J), Fusicoccin R (FC R), 3-Hydroxyfusicoccin J (3-Hydroxy-FC J), Fusicoccin S (FC S), and Phomopsiol toward Lettuce Seeds in the Presence of ()-Abscisic Acid (ABA 10 g/ml) in the Dark (% germination at 25–26 CofLactuca sativa, cv. Olympia) FC J FC R 3-Hydroxy-FC J FC S Phomopsiol ABA Time (hr) (g/ml) (g/ml) (g/ml) (g/ml) (g/ml) (g/ml) None 1 3 10 1 3 10 1 3 10 10 30 100 10 30 100 10 12 51 90 92 0 6 92 36 85 92 0 0 0 0 0 0 0 38 24 96 96 98 19 65 98 98 98 98 0 0 0 0 0 0 0 100 36 100 100 100 59 98 100 100 100 100 0 0 0 0 0 0 0 100 48 100 100 100 73 100 100 100 100 100 0 0 0 0 0 0 0 100

Seed germination-stimulating assay of fusicoccins R Their stimulating effects on lettuce seed (Lactuca sativa, and S, and phomopsiol.15) The seed germination-stim- cv. Olympia) germination are shown in Table 2. ulating activity of fusicoccins R and S, and of phomopsiol together with 3-hydroxy-FC J was exam- Results and Discussion ined in the presence of ()-abscisic acid (10 g/ml) by a method similar to that used in our previous study. P. amygdali Niigata 2-A was cultured in the sta- 1128 N. TAJIMA et al.

17

OH

8 H H H H

5 9 H 4 6 OH H HO H 3 11 CH 1 2 18 16 HO

Fig. 2. Observations of DIF-NOEs (!) in Phomopsiol. tionary mode for 21 days at 25 C in 500-ml flasks (80 only between 17-H and 9-H/4-H ( 1.66), and 18-H flasks), each containing rice bran (26 g), wheat bran and 9-H, but also between 6-H and 8-H/5-H ( 1.90), (13 g) and deionized water (40 ml). An aq. acetone and 16-H ( 3.40) and 5-H/4-H ( 1.79) (Fig. 2).8) solution was prepared from the mycelia and media by Therefore, substance A was identified as O-demethyl-3- homogenization in acetone and gave an EtOAc extract epicotylenol and called phomopsiol. We have also (34.5 g) by EtOAc extraction at pH 9.0. This extract was isolated a new diterpene compound from the culture washed with n-hexane and then with a 2:1 mixture of n- filtrate of the F6 strain of Fusicoccum amygdali, which hexane/ether to give the residual EtOAc extract (ca. was identified as phomopsiol in this report. 6.1 g). This residual extract was separated by silica gel Substance B was identified as 3-hydroxy-FC J as the flash chromatography to give 10 fractions. CN-type expected metabolite from this fungus by comparing its 1 13 substances having a hydroxy group at the 3-position in mp, ½D value, and H- and C-NMR data (Table 1) the aglycons were clearly detected in fractions 3, 4 and 9 with those of 3-hydroxy-FC J (Fig. 3).13) Its stereo- by a TLC analysis, using a 0.5% vanillin-conc. H2SO4 chemistry at C-3 has been principally deduced from the spray reagent. Substances A (fine colorless crystal, chemical shifts of the 1H- and 13C-NMR signals.13) 18 mg), B (colorless crystal, 3.3 mg), C (colorless solid, Identification of phomopsiol having the 3-hydroxy 13.9 mg), and D (colorless solid, 6.3 mg) were isolated group from the fungus encouraged us to confirm its C-3 by rechromatography and preparative TLC. stereochemistry. DIF-NOE experiments on substance B The physicochemical properties of substance A were were done by irradiation at 17-H to give a clear NOE similar to those of the O-demethyl derivative15) of signal of 16-H ( 3.40), thus confirming the presence of cotylenol (the CN A aglycon) which had been previ- the 3-hydroxy group in the molecule (Fig. 3). ously prepared by the addition of DL-ethionine (as an The molecular formula of substance C was deter- inhibitor of the O-methylation of the secondary metab- mined as C34H54O12 from its HR-FABMS data; this olites) to the culture of the CN-producing fungus.15) differs from that of 30-deacety-FC A only by one more However, significant differences between them in the oxygen atom. The 1H-NMR spectrum closely resembled 0 ½D and chemical shift values ( þ1:4–1:9 ppm) of that of 3 -deacetyl-FC A. A distinct difference between the 13C-NMR signals (C-1, 5, 16, and 17 in Table 1) them was observed in the absence or presence of the H-3 were apparent. In addition, careful irradiation at the 17- signal ( 2.79 (1H, m) in 30-deacety-FC A) in their NMR H signal of substance A did not produce any DIF-NOE spectra. In addition, the characteristic signal of H-1 ( signals of 16-H.14) These observations suggested that 5.54) in substance C appeared as a doublet (J ¼ 2:4 Hz), substance A was the 3-epimer of O-demethylcotylenol. while this signal ( 5.32) in 30-deacetyl-FC A appeared The stereochemistry of substance A was elucidated by as a triplet (J ¼ 1:8 Hz). These observations suggested DIF-NOE spectrometry, using a 600 MHz NMR spec- that substance C was a 3-hydroxy derivative of 30- trometer: DIF-NOEs in substance A were observed not deacetyl-FC A. The 1H- and 13C-NMR signals of Novel Fusicoccins R and S, and Phomopsiol and Their Germination-stimulating Activity 1129

OH OH HO OH HO OH O O O O

HO OH HO O HO O OAc H H H

OH OH

OH OMe OMe HO HO HO

HO HO OH 3α-Hydroxyfusicoccin J Fusicoccin R (3α-Hydroxy-FC J: Substance B) (FC R: Substance C) H H

OH HO OH O OH (+)-Fusicocca- HO OH HO O 2,10(14)-dien-8β-ol H H

OH HO HO OH Phomopsiol Fusicoccin S (Substance A) (FC S: Substance D)

Fig. 3. Structures of Fusicoccins R and S, 3-Hydroxyfusicoccin J and Phomopsiol, and Possible Biosynthetic Pathways to Fusicoccins R and S from (þ)-Fusicocca-2,10(14)-dien-8-ol of the Common Biosynthetic Intermediate. substance C were fully assigned by the COSY, DEPT J had high activity similar to that of FC J. On the other and HMBC methods (Table 1), clearly supporting its hand, FC S and phomopsiol, these being a 16-O- structure of 30-deacetyl-3-hydroxy-FC A. The -orien- demethylated 3-epi-FC and its aglycon, were inactive at tation of the hydroxy group at the 3 position was a concentration of 100 g/ml. We are planning to assay determined by the NOE results: irradiation of H-17 ( these new CN-type fusicoccins toward acute myeloid 0.83) markedly enhanced the signals of H-16 ( 3.40). leukemia cells after preparing sufficient amounts. The structure of substance C, therefore, was determined The two-biosynthetic pathways giving FC A and 16- to be 30-deacetyl-3-hydroxy-FC A (Fig. 3). This new O-demethyl-3-epi-FC J in their late stage of the non 3- FC was called fusicoccin R (FC R). hydroxylation pathway have been briefly discussed in a Substance D was determined to have the molecular previous paper; these fusicoccins have no 3-hydroxy 7) formula of C26H42O9 based on its HR-FABMS data. Its group in their molecule. We found in this study that P. 1H- and 13C-NMR signals, being fully assigned by the amygdali Niigata 2-A produced FC S of an unexpected COSY, DEPT and HMBC methods (Table 1), closely 3-hydroxyfusicoccin and phomopsiol of its aglycon, resembled those of phomopsiol and FC H (Fig. 1). These together with 3-hydroxyfusicoccins of FC R and 3- observations suggested that substance D was the 3- hydroxy-FC J. The isolation of FC S and phomopsiol hydroxy derivative of 3-epi-FC H. The -orientation of from the fungus suggested the presence of an additional the hydroxy group at the 3-position was confirmed by new 3-hydroxylation route in the 3-hydroxylation the results of the NOE experiments in the same way as pathway of fusicoccin biosynthesis (Fig. 3). Figure 3 those of phomopsiol: DIF-NOEs were observed between illustrates two biosynthetic routes from (þ)-fusicocca- 17-H and 9-H, 18-H and 9-H, 6-H and 8-H/5-H ( 2,10(14)-dien-8-ol4) to FC S as 3-hydroxy fusicoccin 1.78), and 16-H ( 3.26) and 5-H. The structure of and FC R as 3-hydroxyfusicoccin; the 3- and 3- substance D, therefore, was determined to be 3- hydroxyfusicoccins seem to be formed from the corre- hydroxy-3-epi-FC H (Fig. 3). This new FC was called sponding 3,16-epoxides derived from a fusicoccatriene fusicoccin S (FC S). The isolation of phomopsiol, one of of the common intermediate. the fusicoccin aglycons, from the culture filtrate of this fungus was done for the first time, although related Acknowledgments metabolites of (þ)-fusicocca-2,10(14)-diene and its 8- ol have been isolated as biosynthetic intermediates of The authors thank Dr. S. Kanematsu of the Apple FC A from its mycelial extract.4) Research Center, National Institute of Fruit Tree The germination-stimulating activity of the newly Science, for her help in providing the peach Fusicoccum isolated fusicoccins toward lettuce seeds in the presence canker fungi. This work was supported in part by grant- of ()-abscisic acid (10 g/ml) was examined (Table 2). aid (Nos. 15658036 and 12556015 to T. Sassa) for FC R and 3-hydroxy-FC J, these being 16-O-meth- scientific research from the Ministry of Education, ylated fusicoccins, respectively induced the germination Culture, Sports, Science and Technology of Japan. at low concentrations of 1 and 3 g/ml, 3-Hydroxy-FC 1130 N. TAJIMA et al. References 8) Sassa, T., Ooi, T., Nukina, M., Ikeda, M., and Kato, N., Structural confirmation of cotylenin A, a novel fusicoc- 1) Graniti, A., Ballio, A., and Marre`, E., Fusicoccum cane-diterpene glycoside with potent plant growth- (Phomopsis) amygdali. In ‘‘Pathogenesis and Host regulating activity from Cladosporium fungus sp. 501- Specificity in Plant Diseases’’ (‘‘Eukaryotes’’ Vol. 2), 7W. Biosci. Biotechnol. Biochem., 62, 1815–1818 eds. Singh, U. S., Kohmoto, K., and Singh, R. P., (1998). Pergamon Press, London, pp. 103–117 (1994). 9) Sassa, T., Tojyo, T., and Munakata, K., Isolation of a 2) Tajima, N., Kume, H., Kanematsu, S., Kato, N., and new plant growth substance with cytokinin-like activity. Sassa, T., Chemical identification of fusicoccins from a Nature, 227, 379 (1970). Japanese isolate Niigata 2 of peach Fusicoccum canker 10) Sassa, T., Cotylenins, leaf growth substances produced fungus (Phomopsis amygdali) and production of 30- by a fungus: Isolation and characterization of cotylenins deacetylfusicoccin A by the fungus. J. Pesticide Sci., 27, A and B. Agric. Biol. Chem., 35, 1415–1418 (1971). 64–67 (2002). 11) Honma, H., Ishii, Y., Sassa, T., and Asahi, K., Treatment 3) Ballio, A., Brufani, M., Casinovi, C. G., Cerrini, S., of human promyelocytic leukemia in the SCID mouse Fedeli, W., Pellicciari, R., Santurbano, B., and Vaciago, model with cotylenin A, an inducer of myelomonocytic A., The structure of fusicoccin A. Experientia, 24, 631– differentiation of leukemia cells. Leuk. Res., 11, 1019– 635 (1968). 1025 (2003). 4) Sassa, T., Zhang, C.-S., Sato, M., Tajima, N., Kato, N., 12) Honma, Y., Ishii, Y., Tamamoto-Yamaguchi, Y., Sassa, 2 and Mori, A., Conversion of [19- H2]fusicocca- T., and Asahi, A., Cotylenin A, a differentiation- 2,10(14)-diene into its 8-ol and fusicoccins by Pho- inducing agent, has an antitumor effect on human non- mopsis (Fusicoccum) amygdali. Tetrahedron Lett., 41, small cell lung carcinoma cells in nude mice. Cancer 2401–2404 (2000). Res., 63, 3659–3666 (2003). 5) Bottalico, A., Graniti, A., and Lerario, P., Further 13) Ballio, A., Casinovi, C. G., Grandolini, G., Marta, M., investigation on the biological activity of some fusicoc- and Randazzo, G., 19-Deoxy-3-hydroxydideacetyl- cins and cotylenins. Phytopath. Medit., 17, 127–134 fusicoccin. Gazzetta Chimica Italiana, 105, 1325–1328 (1978). (1975). 6) Oecking, C., Wurtele, M., Ottmann, C. J., and 14) Sassa, T., Zhang, C.-S., Tajima, N., and Kato, N., 16-O- Wittinghofer, A., Structural view of a fungal toxin Demethyl fusicoccin J and its 3-epimer from Fusico- acting on a 14-3-3 regulatory complex. EMBO J., 22, ccum amygdali, and their seed germination-stimulating 987–994 (2003). activity in the presence of abscisic acid. Biosci. 7) Sassa, T., Tajima, N., Sato, M., Takahashi, A., and Kato, Biotechnol. Biochem., 63, 951–954 (1999). N., Fusicoccins P and Q, and 3-epifusicoccins H and Q, 15) Sassa, T., Sakata, Y., Nukina, M., and Ikeda, M., new polar fusicoccins from isolate Niigata 2-A of a Germination-stimulating activity and chemical structure peach Fusicoccum canker fungus. Biosci. Biotechnol. of cotylenin. J. Chem. Soc. Jpn. (in Japanese), 895–897 Biochem., 66, 2356–2361 (2002). (1981).