Phoma medicaginis Isolate Differences Determine Disease Severity and Phytoestrogen Production in Annual Medicago spp.

Phoma black stem and leaf spot disease of annual Medicago spp., caused by Phoma medicaginis, not only can devastate forage and seed yield but can reduce herbage quality by inducing production of phytoestrogens (particularly coumestrol and 4'-O-methylcoumestrol), which can also reduce the ovulation rates of animals grazing infected forage. We determined the consequent phytoestrogen levels on three different annual Medicago species/cultivars (Medicago truncatula cultivar Cyprus, Medicago polymorpha var. brevispina cultivar Serena, and Medicago murex cultivar Zodiac) after inoculation with 35 isolates of P. medicaginis. Across the isolate × cultivar combinations, leaf disease incidence, petiole/stem disease incidence, leaf disease severity, petiole disease severity, and leaf yellowing severity ranged up to 100, 89.4, 100, 58.1, and 61.2%, respectively. Cultivars Cyprus and Serena were the most susceptible and cultivar Zodiac was the most resistant to P. medicaginis. Isolates WAC3653, WAC3658, and WAC4252 produced the most severe disease. Levels of phytoestrogens in stems ranged from 25 to 1,995 mg/kg for coumestrol and from 0 to 418 mg/kg for 4'-O-methylcoumestrol. There was a significant positive relationship of disease incidence and severity parameters with both coumestrol and 4'-O-methylcoumestrol contents, as noted across individual cultivars and across the three cultivars overall, where r = 0.39 and 0.37 for coumestrol and 4'-O-methylcoumestrol, respectively (P < 0.05). Although cultivar Serena was most susceptible to P. medicaginis and produced the highest levels of phytoestrogens in the presence of P. medicaginis, cultivar Zodiac contained the highest levels of phytoestrogens in comparison with other cultivars in the absence of P. medicaginis. There was a 15-fold increase in coumestrol in cultivar Serena but only a 7-fold increase in cultivar Zodiac from infection of P. medicaginis. The study highlights that the intrinsic ability of a particular cultivar to produce phytoestrogens in the absence of the pathogen, and its comparative ability to produce phytoestrogens in the presence of the P. medicaginis, are both important and highly relevant to developing new annual Medicago spp. cultivars that offer improved disease resistance and better animal reproductive outcomes.

Structure-Function Analysis of SMAX1 Reveals Domains That Mediate Its Karrikin-Induced Proteolysis and Interaction with the Receptor KAI2.

Karrikins (KARs) are butenolides found in smoke that can influence germination and seedling development of many plants. The KAR signaling mechanism is hypothesized to be very similar to that of the plant hormone strigolactone (SL). Both pathways require the F-box protein MORE AXILLARY GROWTH2 (MAX2), and other core signaling components have shared ancestry. Putatively, KAR activates the receptor KARRIKIN INSENSITIVE2 (KAI2), triggering its association with the E3 ubiquitin ligase complex SCFMAX2 and downstream targets SUPPRESSOR OF MAX2 1 (SMAX1) and SMAX1-LIKE2 (SMXL2). Polyubiquitination and proteolysis of SMAX1 and SMXL2 then enable growth responses to KAR. However, many of the assumptions of this model have not been demonstrated. Therefore, we investigated the posttranslational regulation of SMAX1 from the model plant Arabidopsis (Arabidopsis thaliana). We find evidence that SMAX1 is degraded by KAI2-SCFMAX2 but is also subject to MAX2-independent turnover. We identify SMAX1 domains that are responsible for its nuclear localization, KAR-induced degradation, association with KAI2, and ability to interact with other SMXL proteins. KAI2 undergoes MAX2-independent degradation after KAR treatment, which we propose results from its association with SMAX1 and SMXL2. Finally, we discover an SMXL domain that mediates receptor-target interaction preferences in KAR and SL signaling, laying the foundation for understanding how these highly similar pathways evolved to fulfill different roles.

(Methylthio)phenol semiochemicals are exploited by deceptive orchids as sexual attractants for Campylothynnus thynnine wasps.

Until recently, (methylthio)phenols as natural products had only been reported from bacteria. Now, four representatives of this class of sulfurous aromatic compounds have been discovered as semiochemicals in the orchid Caladenia crebra, which secures pollination by sexual deception. In this case, field bioassays confirmed that a 10:1 blend of 2-(methylthio)benzene-1,4-diol (1) and 4-hydroxy-3-(methylthio)benzaldehyde (2) sexually attracts the male thynnine wasp Campylothynnus flavopictus (Tiphiidae:Thynnineae), the exclusive pollinator of C. crebra. Here we show with field bioassays that another undescribed species of Campylothynnus (sp. A) is strongly sexually attracted to a 1:1 blend of compounds 1 and 2, which elicits very high attempted copulation rates (88%). We also confirm that this Campylothynnus species is a pollinator of Caladenia attingens subsp. attingens. Chemical analysis of the flowers of this orchid revealed two (methylthio)phenols, compound 2 and 2-(methylthio)phenol (3), as candidate semiochemicals involved in pollinator attraction. Thus, (methylthio)phenols are likely to be more widely used than presently known. The confirmation of this Campylothynnus as a pollinator of C. attingens subsp. attingens at our study sites was unexpected, since elsewhere this orchid is pollinated by a different thynnine wasp (Thynnoides sp). In general, sexually deceptive Caladenia only use a single species of pollinator, and as such, this unusual case may offer a tractable study system for understanding the chemical basis of pollinator switching in sexually deceptive orchids.

Antibacterial compounds from the Australian native plant Eremophila glabra.

Recent reports of Eremophila glabra (R.Br.) Ostenf. (Scrophulariaceae) displaying antibacterial activity has led us to investigate the bioactive secondary metabolites responsible for this activity. Bioassay-directed fractionation of solvent extracts prepared from the leaves of E. glabra led to the isolation of seven serrulatane diterpenes, three flavonoids and the caffeoyl ester disaccharide verbascoside. Among these, four serrulatanes, namely 18-acetoxy-8, 20-dihydroxyserrulat-14-en-19-oic acid (14), 18,20-diacetoxy-8-hydroxyserrulat-14-en-19-oic acid (16), 8,18,20-triacetoxyserrulat-14-en-19-oic acid (17) and 18-acetoxy-8-hydroxyserrulat-14-en-19-oic acid (18) are described for the first time, while 8,20-diacetoxyserrulat-14-en-19-oic acid (3), 8,18,20-trihydroxyserrulat-14-en-19-oic acid (5) and 20-acetoxy-8-hydroxyserrulat-14-en-19-oic acid (19) were previously reported. All three flavonoids hispidulin (12), jaceosidin (13) and cirsimaritin (15) are known but reported for the first time in E. glabra. All compounds were tested in an agar diffusion antimicrobial assay against Staphylococcus aureus (NCTC 10442) and Staphylococcus epidermidis (ATCC 14990). Compounds 12, 13, 17, 18 and 19 exhibited moderate activity, with minimum inhibitory concentrations (MICs) ranging from 32 to 512µg/mL. Compound 19 demonstrated the highest activity against S. epidermidis ATCC 14990 with MIC of 32µg/mL, while 13 demonstrated the highest activity against S. aureus NCTC 10442 with MIC of 128µg/mL.

Karrikins Identified in Biochars Indicate Post-Fire Chemical Cues Can Influence Community Diversity and Plant Development.

BACKGROUND: Karrikins are smoke-derived compounds that provide strong chemical cues to stimulate seed germination and seedling growth. The recent discovery in Arabidopsis that the karrikin perception system may be present throughout angiosperms implies a fundamental plant function. Here, we identify the most potent karrikin, karrikinolide (KAR1), in biochars and determine its role in species unique plant responses. METHODS: Biochars were prepared by three distinct commercial-scale pyrolysis technologies using systematically selected source material and their chemical properties, including karrikinolide, were quantified. Dose-response assays determined the effects of biochar on seed germination for two model species that require karrikinolide to break dormancy (Solanum orbiculatum, Brassica tourneforttii) and on seedling growth using two species that display plasticity to karrikins, biochar and phytotoxins (Lactuca sativa, Lycopersicon esculentum). Multivariate analysis examined relationships between biochar properties and the plant phenotype. FINDINGS AND CONCLUSIONS: Results showed that karrikin abundant biochars stimulated dormant seed germination and seedling growth via mechanisms analogous to post-fire chemical cues. The individual species response was associated with its sensitivity to karrikinolide and inhibitory compounds within the biochars. These findings are critical for understanding why biochar influences community composition and plant physiology uniquely for different species and reaffirms that future pyrolysis technologies promise by-products that concomitantly sequester carbon and enhance plant growth for ecological and broader plant related applications.

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Discovery of pyrazines as pollinator sex pheromones and orchid semiochemicals: implications for the evolution of sexual deception.

Sexually deceptive orchids employ floral volatiles to sexually lure their specific pollinators. How and why this pollination system has evolved independently on multiple continents remains unknown, although preadaptation is considered to have been important. Understanding the chemistry of sexual deception is a crucial first step towards solving this mystery. The combination of gas chromatography-electroantennographic detection (GC-EAD), GC-MS, synthesis and field bioassays allowed us to identify the volatiles involved in the interaction between the orchid Drakaea glyptodon and its sexually attracted male thynnine wasp pollinator, Zaspilothynnus trilobatus. Three alkylpyrazines and one novel hydroxymethyl pyrazine were identified as the sex pheromone of Z. trilobatus and are also used by D. glyptodon for pollinator attraction. Given that our findings revealed a new chemical system for plants, we surveyed widely across representative orchid taxa for the presence of these compounds. With one exception, our chemical survey failed to detect pyrazines in related genera. Collectively, no evidence for preadaptation was found. The chemistry of sexual deception is more diverse than previously known. Our results suggest that evolutionary novelty may have played a key role in the evolution of sexual deception and highlight the value of investigating unusual pollination systems for advancing our understanding of the role of chemistry in evolution.

Exploring the molecular mechanism of karrikins and strigolactones.

Karrikins and strigolactones are novel plant growth regulators that contain similar molecular features, but very little is known about how they elicit responses in plants. A tentative molecular mechanism has previously been proposed involving a Michael-type addition for both compounds. Through structure-activity studies with karrikins, we now propose an alternative mechanism for karrikin and strigolactone mode of action that involves hydrolysis of the butenolide ring.

Solar irradiation of the seed germination stimulant karrikinolide produces two novel head-to-head cage dimers.

Karrikinolide is a naturally derived potent seed germination stimulant that is responsible for triggering the germination of numerous plant species from various habitats around the world. We now report that solar irradiation of karrikinolide yields two novel head-to-head cage photodimers with the formation, stability and bioactivity of both presented herein.

Mycotoxins produced by Fusarium spp. associated with Fusarium head blight of wheat in Western Australia.

An isolated occurrence of Fusarium head blight (FHB) of wheat was detected in the south-west region of Western Australia during the 2003 harvest season. The molecular identity of 23 isolates of Fusarium spp. collected from this region during the FHB outbreak confirmed the associated pathogens to be F. graminearum, F. acuminatum or F. tricinctum. Moreover, the toxicity of their crude extracts from Czapek-Dox liquid broth and millet seed cultures to brine shrimp (Artemia franciscana) was associated with high mortality levels. The main mycotoxins detected were type B trichothecenes (deoxynivalenol and 3-acetyldeoxynivalenol), enniatins, chlamydosporol and zearalenone. This study is the first report on the mycotoxin profiles of Fusarium spp. associated with FHB of wheat in Western Australia. This study highlights the need for monitoring not just for the presence of the specific Fusarium spp. present in any affected grain but also for their potential mycotoxin and other toxic secondary metabolites.

Structure-activity relationship of karrikin germination stimulants.

Karrikins (2H-furo[2,3-c]pyran-2-ones) are potent smoke-derived germination promoters for a diverse range of plant species but, to date, their mode of action remains unknown. This paper reports the structure-activity relationship of numerous karrikin analogues to increase understanding of the key structural features of the molecule that are required for biological activity. The results demonstrate that modification at the C5 position is preferred over modification at the C3, C4, or C7 positions for retaining the highest bioactivity.

Identification of alkyl substituted 2H-furo[2,3-c]pyran-2-ones as germination stimulants present in smoke.

The butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), is a major compound in smoke responsible for promoting the seed germination of a wide range of plant species. We now report the structure of five alkyl substituted variants of 1 that are also present in smoke. The concentrations of these analogues, as well as that of 1, in a typical smoke-water solution have been determined using high-performance liquid chromatography (HPLC) purification followed by gas chromatography-mass spectrometry (GC-MS) analysis. The analogue, 3,5-dimethyl-2H-furo[2,3-c]pyran-2-one (3), was identified at levels that indicate that it is a contributor to the overall germination-promoting activity of crude smoke extracts.

Preparation of 2H-furo[2,3-c]pyran-2-one derivatives and evaluation of their germination-promoting activity.

The butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), has recently been identified as the germination stimulant present in smoke that promotes the germination of seeds from a wide range of plant species. In this paper, we describe the preparation of a number of analogues of 1 and compare their efficacy in promoting seed germination of three highly smoke-responsive plant species, Lactuca sativa L. cv. Grand Rapids (Asteraceae), Emmenanthe penduliflora Benth. (Hydrophyllaceae), and Solanum orbiculatum Poir. (Solanaceae). The results show that the methyl substituent at C-3 in 1 is important for germination-promoting activity while substitution at C-7 reduces activity. In contrast, bioactivity is mostly retained with analogues substituted at C-4 or C-5.