Plant Pathogen Botrytis Cinerea Research Articles

Differences in Frequency of Transposable Elements Presence in Botrytis cinerea Populations from Several Hosts in Greece

This study was conducted primarily to investigate the presence and frequency distribution of the transposable elements Boty and Flipper in populations of the necrotroph plant pathogen Botrytis cinerea in Greece. In total, 334 isolates were collected from diseased grape, strawberry, tomato, cucumber, kiwifruit, and apple fruit during 2009. The presence of the two transposable elements was based on polymerase chain reaction detection. Results showed that all the sampled hosts occurred in sympatry, with four possible different genotypes (transposa type carrying both transposable elements, Boty type carrying only the Boty element, Flipper type carrying only the Flipper element, and vacuma type carrying neither transposable element). Marked differences in genotype frequencies among populations were observed. In tomato, cucumber, grape, and strawberry, transposa isolates carrying both elements were predominant in the populations whereas, in kiwifruit and apple fruit populations, the vacuma isolates were prevailing. Furthermore, in kiwi and apple fruit populations, high frequencies of Flipper-type isolates were observed. In an attempt to explain the observed predominance of vacuma isolates in kiwifruit populations, the mycelial growth rate of a set of vacuma isolates was compared with the mycelial growth rate of a set of transposa isolates at three different temperatures (0, 10, and 20°C). The same set of isolates was used to compare pathogenicity of isolates on wound-inoculated kiwifruit incubated at two different temperatures (0 and 20°C), in terms of disease incidence and disease severity. In addition, the selected isolates were used to compare their ability in causing latent infections on kiwifruit in the field. The results showed that vacuma and transposa isolates had similar mycelial growth rates at the limiting temperatures of 0 and 10°C, while vacuma isolates grew faster at the optimum temperature of 20°C. Similarly, there was no significant difference regarding pathogenicity on kiwifruit between transposa and vacuma isolates. However, artificial inoculations conducted on blossoms in the field showed that vacuma isolates caused significantly higher incidence of latent infections.

Cassia oil for controlling plant and human pathogens on fresh strawberries

The inhibitory effects of cassia oil on the human pathogen Escherichia coli serotype O157:H7 and the plant pathogen Botrytis cinerea were tested in vitro at different concentrations (200–800 ppm). Cassia oil exhibited antibacterial and antifungal activity against both pathogens. Cassia oil at 400–800 ppm inhibited the growth of E. coli O157:H7 in vitro and on the surface of treated strawberries. Cassia oil also completely inhibited the growth of B. cinerea at 400–800 ppm. Spore germination and germ tube elongation of the pathogens in potato dextrose broth were strongly inhibited in the presence of 100 ppm cassia oil. Cassia oil at all concentrations reduced the percentage of decayed strawberries. Experiments on reducing the development of natural decay in strawberries gave similar results. None of the quality parameters tested (colour, total soluble solids, pH, total acidity and ascorbic acid) was affected by cassia oil treatment. Storage experiments on strawberry showed that the percentage weight loss was reduced by cassia oil treatment. Hence, cassia oil could be an alternative to synthetic chemicals for controlling human and plant pathogens on fruits such as strawberries during postharvest and storage.

Regiolone and Isosclerone, Two Enantiomeric Phytotoxic Naphthalenone Pentaketides: Computational Assignment of Absolute Configuration and Its Relationship with Phytotoxic Activity

AbstractThe absolute configurations of regiolone and isosclerone, two enantiomeric bioactive naphthalenone pentaketides produced by fungi and plants, have been unambiguously assigned by ab initio computational prediction of their theoretical optical rotatory powers and electronic circular dichroism spectra. Isosclerone is produced by the plant pathogen Botrytis cinerea, whereas regiolone is produced by B. fabae. The phytotoxic activities of the two compounds were tested for comparison on faba bean (host of both pathogens) and vine plants (host of only B. cinerea); the (R) configuration at C‐4 was found to be a fundamental structural feature for bioactivity.

Antifungal activity of volatile metabolites emitted by mycelial cultures of saprophytic fungi

The antifungal activity of molecular identified Chilean saprobiontic fungi, Trichoderma viride, Schizophyllum commune and Trametes versicolor, on the fungal plant pathogens Botrytis cinerea and Fusarium oxysporum, and the saprotrophic mould Mucor miehei was investigated using two types of inhibition bioassay: (1) bi-compartmented Petri dishes and (2) two Erlenmeyer flasks connected by their upper parts. The chemical composition of volatile organic compounds (VOCs) released by saprobiontic fungi was also investigated using headspace solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC-MS) analysis. Of the saprobiontic fungi evaluated, one isolate of S. commune showed the highest inhibitory activity against B. cinerea and M. miehei, 86.0±5.4 and 99.5±0.5% respectively. The volatile profiles of fungal isolates were shown to contain a different class of compounds. The major components in the headspace of mycelial cultures were 6-pentyl-α -pyrone (T. viride), ethanol and β -bisabolol (S. commune), and a sesquiterpene alcohol (Tr. versicolor). This is the first study reported on the release of VOCs by Chilean native fungi and their antifungal activity wrt. plant pathogenic fungi.

Botrytis cinerea BcNma is involved in apoptotic cell death but not in stress adaptation

Apoptotic-like programmed cell death (PCD) occurs naturally in fungi during development and might also be induced by external conditions. Candidate apoptotic genes have been characterized in several model fungal species but not in plant pathogenic fungi. Here we report on the isolation and characterization of BcNMA, an orthologue of the human pro-apoptotic gene HtrA2 from the plant pathogen Botrytis cinerea. The predicted BcNma protein shows high homology to the previously characterized Nma111p from Saccharomyces cerevisiae and despite some structural differences it complemented the function of Nma111p in Δnma111 mutant strains. BcNMA-over-expression and mutant strains had enhanced or reduced appearance of apoptotic markers, respectively. However there was no difference in growth response of the wild type and BcNMA-transgenic strains to application of various stresses, and the effect on pathogenicity was marginal in both the over-expression and mutant strains. When considered together these results suggest that although BcNma has a pro-apoptotic activity, it is not a major regulator of apoptosis. The protein probably has additional roles that are unrelated to apoptosis, which lead to the pleotrophic phenotype of the transgenic strains and lack of a clear effect on stress adaptation and pathogenicity.

Microsatellite stability in the plant pathogen Botrytis cinerea after exposure to different selective pressures

The stability of microsatellite markers was investigated in the spore-producing fungus Botrytis cinerea exposed to four growth conditions. This knowledge is essential in order to differentiate mutations from genetic exchanges or recombination in population genetics studies. It is also important when using strains from collections that need to be regularly propagated on medium. Successive spore generations of four isolates of the fungus were realised in plates on different agar media: a nutrient-rich medium, a nutrient-poor medium, a medium supplemented with the antibiotic pyrrolnitrin and a medium supplemented with the fungicide iprodione. The stability of nine microsatellite markers was studied by comparing the molecular pattern obtained between the wild type parent strains and the final generations obtained. The results showed that, despite the phenotypic changes observed in some generations, no changes were observed in the allele size at nine microsatellite loci whatever the selective pressure endured by the fungus. This is the first study that reveals long-term stability of microsatellite markers of a spore-producing fungus exposed to different stresses.

Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system

For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the beta-tubulin (tubA) terminator. Recently, it has been demonstrated that this vector contains a 446-bp portion of the B. cinerea argininosuccinate synthase gene (bcass1) rather than the tubA terminator. As argininosuccinate synthase is essential for the production of L-arginine, inadvertent gene silencing of bcass1 may result in partial L-arginine auxotrophy and, indeed, may lead to altered phenotypes in planta. In this article, we report our findings relating to possible problems arising from this incorrect plasmid construction. As an absolute baseline, gene disruption of bcass1 was carried out and generated a strict auxotroph, unable to grow without exogenous arginine supplementation. The knockout displayed an alteration in host range in planta, showing a reduction in pathogenicity on strawberries, French bean leaves and tomatoes, but maintained wild-type growth on grape, which is in accordance with the reported arginine availability in such tissues. Deliberate gene silencing of bcass1 mirrored these effects, with strongly silenced lines showing reduced virulence. The degree of silencing as seen by partial auxotrophy was correlated with an observed reduction in virulence. We also showed that inadvertent silencing of bcass1 is possible when using the pLOB1 vector or derivatives thereof. Partial arginine auxotrophy and concomitant reductions in virulence were triggered in approximately 6% of transformants obtained when expressing enhanced green fluorescent protein, luciferase, monomeric red fluorescent protein or beta-glucuronidase using the pLOB1-based expression system, which inadvertently contains 446 bp of the bcass1 coding sequence. We recommend the testing of transformants obtained using this vector system for arginine auxotrophy in order to provide assurance that any observed effects on the development or virulence are a result of the desired genetic alteration rather than accidental bcass1 silencing.

Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with approximately 1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 A, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (alpha/beta)(8) barrel fold and each pair swapping the 8th alpha-helix. An active site "gating loop" exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH.ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference.

Novel insights into mannitol metabolism in the fungal plant pathogen Botrytis cinerea

In order to redefine the mannitol pathway in the necrotrophic plant pathogen Botrytis cinerea, we used a targeted deletion strategy of genes encoding two proteins of mannitol metabolism, BcMTDH (B. cinerea mannitol dehydrogenase) and BcMPD (B. cinerea mannitol-1-phosphate dehydrogenase). Mobilization of mannitol and quantification of Bcmpd and Bcmtdh gene transcripts during development and osmotic stress confirmed a role for mannitol as a temporary and disposable carbon storage compound. In order to study metabolic fluxes, we followed conversion of labelled hexoses in wild-type and DeltaBcmpd and DeltaBcmtdh mutant strains by in vivo NMR spectroscopy. Our results revealed that glucose and fructose were metabolized via the BcMPD and BcMTDH pathways respectively. The existence of a novel mannitol phosphorylation pathway was also suggested by the NMR investigations. This last finding definitively challenged the existence of the originally postulated mannitol cycle in favour of two simultaneously expressed pathways. Finally, physiological and biochemical studies conducted on double deletion mutants (DeltaBcmpdDeltaBcmtdh) showed that mannitol was still produced despite a complete alteration of both mannitol biosynthesis pathways. This strongly suggests that one or several additional undescribed pathways could participate in mannitol metabolism in B. cinerea.

Biocatalytic preparation and absolute configuration of enantiomerically pure fungistatic anti-2-benzylindane derivatives. Study of the detoxification mechanism by Botrytis cinerea

Enantiomerically pure 2-benzylindane derivatives were prepared using biocatalytic methods and their absolute configuration determined. (1R,2S)-2-Benzylindan-1-ol ((1R,2S)-2) and (S)-2-benzylindan-1-one ((S)-3) were produced by fermenting baker's yeast. Lipase-mediated esterifications and hydrolysis of the corresponding racemic substrates gave rise to the enantiopure compounds (1S,2R)-2-benzylindan-1-ol ((1S,2R)-2) and (1R,2S)-2-benzylindan-1-ol ((1R,2S)-2), respectively. The antifungal activity of these products against two strains of the plant pathogen Botrytis cinerea was tested. The metabolism of anti-(+/-)-2-benzylindan-1-ol (anti-(+/-)-2) by B. cinerea as part of the fungal detoxification mechanism is also described and revealed interesting differences in the genome of both strains.

The Botrytis cinerea aspartic proteinase family

The ascomycete plant pathogen Botrytis cinerea secretes aspartic proteinase (AP) activity. Functional analysis was carried out on five aspartic proteinase genes ( Bcap1-5) reported previously. Single and double mutants lacking these five genes showed neither a reduced secreted proteolytic activity, nor a reduction in virulence and they showed no alteration in sensitivity to antifungal proteins purified from grape juice. Scrutiny of the B. cinerea genome revealed the presence of nine additional Bcap genes, denoted Bcap6-14. The product of the Bcap8 gene was found to constitute up to 23% of the total protein secreted by B. cinerea. Bcap8 -deficient mutants secreted ∼70% less AP activity but were just as virulent as the wild-type strain. Phylogenetic analysis showed that Bcap8 has orthologs in many basidiomycetes but only few ascomycetes including the biocontrol fungus Trichoderma harzanium. Potential functions of the 14 APs in B. cinerea are discussed based on their sequence characteristics, phylogeny and predicted localization.

Characterization of the cell wall of the ubiquitous plant pathogen Botrytis cinerea

The ascomycete Botrytis cinerea is a destructive and ubiquitous plant pathogen and represents a model organism for the study of necrotrophic fungal pathogens. Higher fungi possess a complex and dynamic multilayer cell wall involved in crucial aspects of fungal development, growth and pathogenicity. Plant resistance to microbial pathogens is determined often by the capacity of the plant to recognize molecular patterns associated with the surface of an interacting microbe. Here we report the chemical characterization of cell walls from B. cinerea during axenic growth. Neutral sugars and proteins constituted most of the mass of the B. cinerea cell walls, although chitin and uronic acids were detected. Glucose was the most abundant neutral sugar, but arabinose, galactose, xylose and mannose also were present. Changes in cell wall composition during culture were observed. As the culture developed, protein levels declined, while chitin and neutral sugars increased. Growth of B. cinerea was associated with a remarkable decline in the fraction of its cell wall material that was soluble in hot alkali. These results suggest that the cell wall of B. cinerea undergoes significant modifications during growth, possibly becoming more extensively covalently cross-linked, as a result of aging of mycelia or in response to decreasing nutrient supply or as a consequence of increasing culture density.

Biosynthesis of the Sesquiterpene Botrydial in Botrytis cinerea. Mechanism and Stereochemistry of the Enzymatic Formation of Presilphiperfolan-8β-ol

Presilphiperfolan-8beta-ol synthase, encoded by the BcBOT2 gene from the necrotrophic plant pathogen Botrytis cinerea, catalyzes the multistep cyclization of farnesyl diphosphate (2) to the tricyclic sesquiterpene alcohol presilphiperfolan-8beta-ol (3), the preursor of the phytotoxin botrydial, a strain-dependent fungal virulence factor. Incubation of (1R)-[1-(2)H]farnesyl diphosphate (2b) with recombinant presilphiperfolan-8beta-ol synthase gave exclusively (5R)-[5alpha-(2)H]-3b, while complementary incubation of (1S)-[1-(2)H]FPP (2c) gave (5S)-[5beta-(2)H]-3c. These results established that cyclization of farnesyl diphosphate involves displacement of the diphosphate group from C-1 with net inversion of configuration and ruled out the proposed intermediacy of the cisoid conformer of nerolidyl diphosphate (9) in the cyclization. While not a mandatory intermediate, (3R)-nerolidyl diphosphate was shown to act as a substrate surrogate. Cyclization of [13,13,13-(2)H(3)] farnesyl diphosphate (2d) gave [14,14,14-(2)H(3)]-3d, thereby establishing that electrophilic attack takes place exclusively on the si face of the 12,13-double bond of 2. The combined results provide a detailed picture of the conformation of enzyme-bound farnesyl diphosphate at the active site of presilphiperfolan-8beta-ol synthase.

The pOT and pLOB vector systems: Improving ease of transgene expression in Botrytis cinerea

This paper outlines the construction of a novel vector system comprising interchangeable terminators, as well as a multiple cloning site (MCS), to facilitate the transformation of the fungal plant pathogen Botrytis cinerea. Previous molecular studies on B. cinerea have relied upon the pLOB1 based vector system (controlled by the Aspergillus nidulans oliC promoter and a region reported to be the B. cinerea tubA terminator). Investigations, however, have revealed that, rather than the genuine B. cinerea tubA terminator, the pLOB1 terminator fragment is from another gene locus within the genome. Because previous studies have found that terminators aide in transcript stability, the main aims of this study were to develop and evaluate both vector systems, pOT (controlled by the A. nidulans oliC promoter and A. nidulans trpC terminator) and pLOB, with a range of exogenous genes, including enhanced green fluorescent protein (eGFP), monomeric red fluorescent protein (mRFP), luciferase (LUC) and beta-glucuronidase (GUS). Our investigations demonstrate that pLOB and pOT based vectors are capable of expressing all four reporter genes and may be applied to future molecular studies on B. cinerea and other related ascomycetes. Additionally, this is the first reported expression of mRFP and LUC in B. cinerea.

Chemical Composition and Antifungal Activity of Arnica longifolia, Aster hesperius, and Chrysothamnus nauseosus Essential Oils

Essential oils from three different Asteraceae obtained by hydrodistillation of aerial parts were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). Main compounds obtained from each taxon were found as follows: Arnica longifolia carvacrol 37.3%, alpha-bisabolol 8.2%; Aster hesperius hexadecanoic acid 29.6%, carvacrol 15.2%; and Chrysothamnus nauseosus var. nauseosus beta-phellandrene 22.8% and beta-pinene 19.8%. Essential oils were also evaluated for their antimalarial and antimicrobial activity against human pathogens, and antifungal activities against plant pathogens. No antimalarial and antimicrobial activities against human pathogens were observed. Direct bioautography demonstrated antifungal activity of the essential oils obtained from three Asteraceae taxa and two pure compounds, carvacrol and beta-bisabolol, to the plant pathogens Colletotrichum acutatum, C. fragariae and C. gloeosporioides. Subsequent evaluation of antifungal compounds using a 96-well micro-dilution broth assay indicated that alpha-bisabolol showed weak growth inhibition of the plant pathogen Botrytis cinerea after 72 h.

Anti-fungal action of bioconverted eicosapentaenoic acid (bEPA) against plant pathogens

Bioconverted eicosapentaenoic acid (bEPA), obtained from Pseudomonas aeruginosa PR3, was assessed for its in vitro and in vivo anti-fungal potential. Mycelial growth inhibition of the tested plant pathogens ( Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum, Fusarium solani, Phyptophthora capsici, Sclerotinia sclerotiorum and Colletotrichum capsici) was measured in vitro. bEPA at the concentration of 5 μl/ml inhibited 52–60% fungal mycelial growth for all of the plant pathogens in vitro except S. sclerotiorum. Minimum inhibitory concentrations (MICs) of bEPA were found in the range of 250–500 μg/ml. Also, bEPA had a detrimental effect on spore germination for all the tested plant pathogens. Three plant pathogenic fungi ( F. oxysporum, P. capsici and C. capsici) were subjected to an in vivo anti-fungal screening. bEPA at the initial concentration of 3000 μg/ml had a 100% anti-fungal effect against all of the tested plant pathogens. Concentrations of bEPA corresponding to 1500, 500 and 300 μg/ml were applied to the plants and revealed promising anti-fungal effects, supporting bEPA as a potential anti-fungal agent.

The Antifungal Activity of Widdrol and Its Biotransformation by Colletotrichum gloeosporioides (penz.) Penz. & Sacc. and Botrytis cinerea Pers.: Fr.

Widdrol (1) was tested against the necrotrophic plant pathogens Botrytis cinerea and Colletotrichum gloeosporioides. While 1 was found to be inactive against C. gloeosporioides, it showed a selective and effective control of B. cinerea, significantly inhibiting the mycelial growth of the fungus at concentrations of 100 ppm and above. In addition, the biotransformation of 1 by both fungi was studied. Incubation with C. gloeosporioides and B. cinerea afforded four and one biotransformation products (2-6), respectively. Biotransformation with C. gloeosporioides was highly regioselective, yielding for the most part oxidation products at C-10: 10-oxowiddrol (2), 10beta-hydroxywiddrol (3), 10alpha-hydroxywiddrol (4), and 14alpha-hydroxywiddrol (5). The structures of all products were determined on the basis of their spectroscopic data, including coupling constants, two-dimensional NMR analysis (heteronuclear multiple quantum coherence, heteronuclear multiple bond correlation, and nuclear Overhauser enhancement spectroscopy), and nuclear Overhauser effect. The biotransformation products were then tested against B. cinerea and found to be inactive. These results shed further light on the structural modifications, which may be necessary to develop selective fungal control agents against B. cinerea.

A Class III Histidine Kinase Acts as a Novel Virulence Factor in Botrytis cinerea

Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.

Antifungal Activity of Thiophenes from Echinops ritro

Extracts from 30 plants of the Greek flora were evaluated for their antifungal activity using direct bioautography assays with three Colletotrichum species. Among the bioactive extracts, the dichloromethane extract of the radix of Echinops ritro (Asteraceae) was the most potent. Bioassay-guided fractionation of this extract led to the isolation of eight thiophenes. Antifungal activities of isolated compounds together with a previously isolated thiophene from Echinops transiliensis were first evaluated by bioautography and subsequently evaluated in greater detail using a broth microdilution assay against plant pathogens Colletotrichum acutatum, Colletotrichum fragariae, Colletotrichum gloeosporioides, Botrytis cinerea, Fusarium oxysporum, Phomopsis viticola, and Phomopsis obscurans. 5'-(3-Buten-1-ynyl)-2,2'-bithiophen (1), alpha-terthienyl (2), and 2-[pent-1,3-diynyl]-5-[4-hydroxybut-1-ynyl]thiophene (5) at 3 and 30 microM were active against all three Colletotrichum species, F. oxysporum, P. viticola, and P. obscurans.

Antifungal Activity and Biotransformation of Diisophorone by Botrytis cinerea

Diisophorone (1) was tested against two strains of the necrotrophic plant pathogen Botrytis cinerea. Fungal sensitivity varied according to the strain. B. cinera 2100 was more sensitive than B. cinereaUCA992: its mycelial growth was significantly inhibited at concentrations of 50 ppm and above. Although diisophorone (1) showed an effective control of B. cinerea, a detoxification mechanism was present. The detoxification of racemic diisophorone (1) by B. cinerea was investigated. Incubation with two strains of B. cinerea gave one and four biotransformation products (2-5), respectively. Their structures were established as the known 8beta-hydroxydiisophorone (2), 6alpha-hydroxydiisophorone (3), 6beta-hydroxydiisophorone (4) and 8beta,14beta-dihydroxydiisophorone (5) on the basis of their spectroscopic data, including two-dimensional NMR analysis [heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC), and nuclear Overhauser enhancement spectroscopy (NOESY)] and an X-ray crystallographic study.