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Local phytochemical response of Musa acuminata × balbisiana Colla cv. ‘Bluggoe’ (ABB) to colonization by Sternorrhyncha
Phytochemistry xxx (2016) 1e7
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Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to colonization by Sternorrhyncha € lscher a, b, *, Antje Vollrath c, d, Marco Kai e, 1, Dirk Ho Suganthaguntalam Dhakshinamoorthy f, 2, Riya C. Menezes e, Ales Svatos e, Ulrich S. Schubert c, d, Andreas Buerkert b, Bernd Schneider a, ** €ll-Str. 8, 07745 Jena, Germany Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology, Hans-Kno Organic Plant Production and Agroecosystems Research in the Tropics and Subtropics (OPATS), University of Kassel, Steinstr. 19, 37213 Witzenhausen, Germany c Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstr. 10, 07743 Jena, Germany d Jena Center of Soft Matter (JCSM), Friedrich Schiller University Jena, Humboldtstr. 10, 07743 Jena, Germany e €ll-Str. 8, 07745 Jena, Germany Research Group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology, Hans-Kno f Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Faculty of Bioscience Engineering, University of Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2016 Received in revised form 10 October 2016 Accepted 12 October 2016 Available online xxx
The interaction of two Sternorrhyncha species, the banana aphid (Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae, Aphidinae)), vector of the banana bunchy top virus (BBTV), and the latania scale (Hemiberlesia lataniae Signoret (Hemiptera: Diaspididae, Diaspidinae)) with Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ (Musaceae) was investigated by a combination of conventional and spatially resolved analytical techniques, 1H NMR, UHPLC-MS, and matrix-free UV-laser desorption/ionization MS imaging. After infestation, the feeding sites of P. nigronervosa on the pseudostem and the exocarp of banana fruit developed a red tinge, in which tissue-specific accumulations of phenylphenalenones were discovered. Phenylphenalenones were also detected in the black mats of sooty molds growing on the banana aphid exudates and in the dorsal scales of H. lataniae. This suggests that although these secondary metabolites play a role in the reaction of banana plants towards attack by sucking insects, an aphid and an armored scale have established mechanisms to exude these metabolites before they deploy their deleterious effect. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Hemiberlesia lataniae Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ Musaceae Pentalonia nigronervosa Aphididae Diaspididae Mass spectrometric imaging Phenylphenalenones Plant-pathogen interaction
1. Introduction Bananas and plantains (Musa spp.) are important food crops in tropical and subtropical areas of the world. In 2013 over 156 million t
* Corresponding author. Organic Plant Production and Agroecosystems Research in the Tropics and Subtropics (OPATS), University of Kassel, Steinstr. 19, 37213 Witzenhausen, Germany. ** Corresponding author. € lscher), [email protected] E-mail addresses: [email protected] (D. Ho (B. Schneider). 1 Present address: Institute of Biological Science, Department of Biochemistry, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany. 2 Present address: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive NW, Atlanta, GA 30332-0100, USA.
bananas and plantains were harvested (FAOSTAT, 2013). These fruits are major staples for more than 400 million people particularly in India, China and many African countries (FAOSTAT, 2013). Fungi, insects, plant-parasitic nematodes and viruses can have serious negative effects on banana yields. The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) is a known vector of banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus; Fauquet et al., 2005), one of the diseases most heavily affecting banana yields in the Pacific, South and South-East Asia and parts of Africa (Dale, 1987; Kagy et al., 2001; Kumar et al., 2011). P. nigronervosa was first described in the 19th century on the union (Coquerel, 1859), although the aphid likely origisland of Re inated in Southeast Asia, as did the genus Musa (Waterhouse, 1987; Robinson, 1996). P. nigronervosa is now widely distributed in the
http://dx.doi.org/10.1016/j.phytochem.2016.10.007 0031-9422/© 2016 Elsevier Ltd. All rights reserved.
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
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€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
tropics and in greenhouses in Europe and North America (Blackman and Eastop, 1984) but appears to specifically affect plants of the genus Musa (Foottit et al., 2010). BBTV is the causal agent of banana bunchy top disease (BBTD) and the banana aphid transmits the virus in a circulative, nonpropagative manner (Magee, 1927; Hu et al., 1996). While the Sigatoka disease complex (caused by Mycosphaerella spp. Johanson, Mycosphaerellaceae) may cause yield losses of up to 30e50% (Mobambo et al., 1993), plants infected gi et al., 1995). with BBTV may become completely unproductive (Sa The movement of infected material is thought to spread the virus over long distances and, once established, the disease is difficult to manage and eradicate (Banerjee et al., 2014). Recent publications addressing the interaction between the banana aphid, BBTV and banana plants have focused on strategies for controlling BBTV (Robson et al., 2006; Hooks et al., 2009) and the development of transgenic banana germplasm to manage the virus (Elayabalan et al., 2013). BBTV is known to infect a range of Musa species, including M. coccinea Andrews (Musaceae), M. ornata Roxb. (Musaceae), M. balbisiana Colla (Musaceae) and M. acuminata ssp. zebrina (Van Houtte ex Planch) Nasution (Musaceae) (Ploetz et al., 2015). In our greenhouses, banana aphid colonies were discovered on red regions of the pseudostem of Musa acuminata balbisiana Colla (ABB group) ‘Bluggoe’ (Musaceae) (BG). BG is a popular starchy banana primarily used for cooking, is grown in many countries and recognized to be resistant to drought and Black and Yellow Sigatoka disease but susceptible to Panama disease (Jones, 1999). In the greenhouse of the University of Kassel in Witzenhausen (Germany), some areas between pseudostem and the base of leaf petioles were colonized by dark pigmented epifoliar fungi (sooty molds) which are reported to take no nutrients or water from the host plants (Hughes, 1976), are not known to induce phytoalexin responses by host plants, but can negatively affect plant growth by reducing the rate of photosynthesis. In the greenhouse of the Max Planck Institute for Chemical Ecology (MPI-CE) in Jena (Germany), an additional sucking insect, latania scale (Hemiberlesia lataniae Signoret, Diaspididae) was observed to colonize Musa spp. H. lataniae belongs to the family of armored scales (Diaspididae), a group of plant parasitic arthropods including some of the most damaging pests of perennial crops and ornamentals (Beardsley and Gonzalez, 1975; Miller et al., 2005). The latania scale is highly polyphagous and occurs throughout the world in tropical to temperate zones (Miller and Davidson, 2005; Normark and Johnson, 2011; CABI, 2015). In the case of bananas, some species of armored scales (Hemiberlesia musae Takagi & Yamamoto (Diaspididae) and Hemiberlesia ocellata Takagi & Yamamoto (Diaspididae)) have been observed on infested imported banana fruits (Takagi and Yamamoto, 1974). The coconut scale, Aspidiotus destructor Signoret (Diaspididae), was reported to cause heavy infestations resulting in yellowing and necrosis of leaves of banana in Hawaii (Wright and Diez, 2005). Many plant secondary metabolites possess insecticidal activities. Deterrent effects of plant metabolites such as the isoflavone genistein and the flavone luteolin on phloem-sucking insects such as the pea aphid (Acyrthosiphon pisum Harris (Aphididae)) prompted breeding of plants resistant to phloem-feeding insects (Goławska and Łukasik, 2012). Phenylphenalenones play a major € lscher et al., 2014). In role in the banana plant defense system (Ho Musa spp., the formation of phenylphenalenone-related compounds has been elicited in the peels of fruit by Colletotrichum musae (Berk. & M.A. Curtis) Arx (Glomerellaceae) (Anthracnose disease), in leaves by Mycosphaerella fijiensis Morelet (Mycosphaerellaceae) (Black Sigatoka leaf streak disease), and in roots and rhizomes by Fusarium oxysporum f. sp. cubense (E.F.Sm.) W.C.Snyder & H.N.Hansen (Nectriaceae) (Panama disease) (Luis et al., 1994; Ot alvaro et al., 2007; Hirai et al., 1994). A combination of
conventional and spatially resolved analytical techniques (NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging (LDI-MSI), and Raman microspectroscopy) was applied to identify and locate phenylphenalenones in lesions caused by the burrowing nematode Radopholus similis (Cobb) Thorne (Pratylenchidae) in the roots of banana cultivars differing in €lscher et al., 2014). The study of this susceptibility to R. similis (Ho interaction provided evidence for the local induction of phenylphenalenone-type secondary metabolites in response to R. similis infection in Musa spp. The banana-nematode interaction demonstrated the effects of phenylphenalenone-type compounds on an important pathogen of banana plants. The present study reports the phenylphenalenones occurring in the red regions of the pseudostem and the fruit exocarp of BG that were colonized by P. nigronervosa, in the black mats of dark pigmented epifoliar fungi (sooty molds) and the dorsal scales of H. lataniae. 2. Results and discussion 2.1. Pseudostem and banana peel discoloration e banana aphids, sooty molds and armored scales In the greenhouse, BG plants were colonized with P. nigronervosa (Fig. 1A) between pseudostem and petiole leaf and on banana fruits. The pseudostems of one year old plants and four week old banana fruits were monitored for aphid damage. The development of a red tinge was observed on the surface of aphidcolonized plant tissues (Fig. 1BeE), while aerial, aphid free areas of the banana plants appeared healthy upon visual inspection. As leaf petioles grew away from the pseudostem, the red-colored areas which were also covered by aphid honeydew were exposed to epifoliar fungi and sooty mold mats developed on them (Fig. 1F). In the MPI-CE greenhouse, banana plants were additionally colonized by armored scales (H. lataniae). The latania scales caused dark red regions on the banana pseudostems (Fig. 1G and H). It appears that the observed coloration is the response of banana plants to attack by the banana aphid and the armored scale, respectively. Armored scale infestations have been reported to cause leaf chlorosis and the development of colored spots on different plant species such as coconuts, other palms and pineapples (Beardsley and Gonzalez, 1975). Olive scales (Parlatoria oleae Colvee (Diaspididae)) induce purple spots on the green olives turning straw colored as the fruits mature (McKenzie, 1952). Recently Hill et al. (2011) showed the development of bowl shaped wound periderm encircling latania scales on the canes of some genotypes of kiwifruit (Actinidia chinensis L. (Actinidiaceae)). They report that an extensive deposition of phenolic compounds within the suberized/lignified wound periderm cells and cortical cells beneath and around the insect prevents the scales stylet from reaching unmodified parenchyma tissue on which this diaspidid scale could feed, ultimately leading to the death of the scale. As opposed to the lethal response of some kiwifruit genotypes on latania scales on the canes, the formation of a red tinge on the pseudostem and the fruit exocarp of BG did not kill the banana aphid or the latania scale. Nonetheless, the eurymerous behavior of latania scales allows the survival of members of this species on kiwifruit plants. 2.2. Isolation, identification and function of plant secondary metabolites The red colored areas of the pseudostem from BG and the fruit exocarp of BG were manually dissected using scissors. Green (pseudostem) and yellow (exocarp) healthy control material was additionally checked under a stereo microscope for the appearance
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
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Fig. 1. Photos of sucking insects and their damage on BG tissues. (A) An adult banana aphid (P. nigronervosa) on red-colored pseudostem material of BG. (B, C) Dark red colored regions of the pseudostem of BG colonized by the banana aphid. (D) Red colored areas on the peel of a ripe and a green BG banana fruit, covered with the shed skins of banana aphids (E). (F) Black mats of sooty mold on BG pseudostem. (G) Adult H. lataniae on red colored areas of BG pseudostems and (H) a single female adult latania scale with its membranous ventral scale separating the insect from direct contact with the plant surface and the experimentally removed dorsal scale on the right side of the photo.
of putative small red-colored regions, which were removed with a scalpel and discarded. The mesocarp of the banana fruit was removed from the exocarp using a spoon with rounded edges. The dark pigmented epifoliar sooty molds were removed from the surface of the pseudostem in the same way. A microspoon with rounded edges was used to collect 500 mg dorsal scales of H. lataniae. All samples were separately subjected to extraction using ethanol. Liquid-liquid separation resulted in CHCl3, ethyl acetate and aqueous subfractions. 1H NMR analysis of the CHCl3 subfraction of the P. nigronervosa-colonized red pseudostem material showed signals in the aromatic range of the spectrum while the corresponding fraction obtained from green tissue did not show such signals. The CHCl3 subfraction of the red tissue was then purified by preparative layer chromatography (PLC). The purified compounds were subjected to 1H NMR spectroscopy and four compounds were identified as anigorufone (1), isoanigorufone (2), hydroxyanigorufone (3), and irenolone (4) (Fig. 2; Cooke and Thomas, 1975; Luis et al., 1993, 1999). All the compounds are known as typical metabolites and phytoalexins of Musa species lvaro et al., 2007). (Luis et al., 1994; Hirai et al., 1994; Ota Equal amounts of all the CHCl3-subfractions were subjected to UHPLC-ESI-MS analysis (Fig. 3), with the exception of the CHCl3subfraction of the dorsal scales of H. lataniae. The small levels of phenylphenalenones occurring in the latter sample required the
OH R
O
1R=H 3 R = OH
OH R
O
2R=H 4 R = OH
Fig. 2. Structures of isolated phenylphenalenone-type phytoalexins 1e4. (1) Anigorufone, (2) isoanigorufone, (3) hydroxyanigorufone and (4) irenolone.
Fig. 3. Extracted-ion chromatograms of a UHPLC-ESI-MS analyses of CHCl3-subfractions of ethanolic extracts obtained from red areas of the P. nigronervosa colonized BG pseudostem (red line), from red regions from P. nigronervosa-colonized BG exocarp (blue line), from sooty mold colonizing P. nigronervosa honeydew (brown line), from the dorsal scales of H. lataniae (pink line) and from adults of P. nigronervosa (green line). (1) Anigorufone, (2) isoanigorufone, (3) hydroxyanigorufone and (4) irenolone.
use of a less diluted sample (1:10 instead of 1:100). The phytochemical profiles of the red regions of the BG pseudostem, the dorsal scales of H. lataniae and the sooty mold showed a high similarity concerning the occurrence of the four phenylphenalenones 1e4. A better resolution for the result of the dorsal scales of H. lataniae is shown in Fig. S1 (Supporting information). The amount of anigorufone (1) and isoanigorufone (2) is higher than the amount of the two p-hydroxylated phenylphenalenones (3 and 4). In the red regions of the BG fruit exocarp, hydroxyanigorufone (3) and irenolone (4) are the major compounds in comparison with anigorufone (1) and isoanigorufone (2). These results are
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an excellent example of the capability of different parts of banana plants to respond with different phytochemical profiles to a pest. The spectra of the banana aphid extracts (Fig. 3) as the spectra of the controls showed no evidence of the compounds 1e4. The banana aphids take up substantial amounts of host plant content (Leroy et al., 2011) to extract nitrogen-containing nutrients, constantly exuding honeydew, and presumably not allowing phenylphenalenones to accumulate in their bodies. The proof of phenylphenalenones in the sooty molds was the first hint that phenylphenalenones are transported through the bodies of aphids and, without being markedly metabolized, are exuded into their surroundings. Under the greenhouse conditions at the University of Kassel in Witzenhausen, aphids colonized the area below the Vshape formed by leaf petioles and pseudostems of banana plants. Due to this location, their exudates accumulated on the inner side of the leaf petiole over time. Sooty molds colonized the exudates of aphid colonies which had accumulated over the period of one year. The material which was investigated therefore contained nonvolatile secondary metabolites exuded by the banana aphids over the period of complete leaf expansion (Fig. 3). In contrast, banana aphid extracts were collected from individuals with a maximum lifespan of four weeks, during which time it is presumed that they continuously exude honeydew containing the plant secondary metabolites. The absence of phenylphenalenones in the aphid's body extract is strikingly different from findings of a recent study of different Musa-derived phenylphenalenone-type compounds in the burrowing nematode R. similis. Anigorufone (1), which caused the greatest nematostatic and nematocidal effects among the phenylphenalenones tested, accumulated in the body of the nem€ lscher et al., 2014). Further evidence for a lack of detriatode (Ho mental effects of phenylphenalenones on P. nigronervosa was obtained when we cultivated different bananas varieties in plexiglass containers in the presence of banana aphids; none of the plants in infested containers survived the attack. Obviously phenylphenalenones are ineffective agents against banana aphids because the metabolites passed through these insects without apparently harming them. Thus there was no reason to assay deterrent or toxic effects of phenylphenalenones on the aphids. Different from P. nigronervosa, the exudates of H. lataniae are not distributed within the neighboring space but stored in their dorsal scales. Armored scale insects do not excrete honeydew and lack the filter chamber type of digestive system found in most other Coccoidea (Beardsley and Gonzalez, 1975). It has been shown that the material excreted through the anus is used in the formation of the dorsal scale (Disselkamp, 1954; Beardsley and Gonzalez, 1975). The presence of phenylphenalenones in these dorsal scales indicates that H. lataniae also takes up and exudes these plant secondary metabolites without being noticeably affected by them. Transcriptome analysis of kiwifruit bark in response to latania scales and of banana plants to C. musae, respectively, showed the significant upregulation of transcripts associated with secondary metabolism (Tang et al., 2013; Hill et al., 2015). The occurrence of transcripts specific for enzyme orthologues including phenylalanine ammonia lyase, chalcone synthase and coumarate CoA ligase which can be involved in the biosynthesis of either phenylphenalenones or phenolic compounds (i.e. flavonoids and tannins) can be regarded as further evidence for the role of these secondary metabolites in the pathogen defense response of plants.
can be focused on spots down to 10 mm to allow spatial information of phenylphenalenones in the red-colored pseudostem regions. It revealed the accumulation of secondary metabolites in the aphidcolonized regions. The LDI-MSI images were plotted in FlexImaging software 3.0 after spectra baseline correction, hotspot removal (by suppressing 1% of the most intense pixels), and edge-preserving image-denoising to reduce the pixel-to-pixel variation. The m/zvalues of the discovered plant secondary metabolites in the red pseudostem region are consistent with the four phenylphenalenones (Fig. 2) from the phytochemical investigation of the aphid-infested material. A signal for anigorufone (1) and isoanigorufone (2) (m/z 271, [MH]) was found in this area and the m/z 287 ([MH]) value detected within the red colored pseudostem region was assigned to [MH] of hydroxyanigorufone (3) and irenolone (4) (Fig. 4). The green aerial parts and the red pseudostem regions colonized by the banana aphids were strikingly different from each other with regard to the presence of phenylphenalenones, both in the phytochemical extractions and identifications as well as in the LDI-MSI investigations. The formation of phenylphenalenone-type compounds by bananas has been reported as a response to infection with R. similis (burrowing nematode), C. musae (Anthracnose), M. fijiensis (Black Sigatoka) and F. oxysporum f. sp. cubense (Panama disease; Luis lvaro et al., 2007; Hirai et al., 1994; Ho €lscher et al., et al., 1994; Ota 2014; Hidalgo et al., 2016). The present phytochemical investigations provide evidence for the local induction of phenylphenalenones in Musa ABB cv. ‘Bluggoe’ in response to the banana aphid and the latania scale and extend the number of banana pathogens that elicit the site-specific formation of phenylphenalenone-related compounds. Four typical phytoalexins of the phenylphenalenone-type were shown by NMR and LDI-MSI analyses to be present in the affected pseudostem tissues of Musa plants. Anigorufone (1) was the most abundant phenylphenalenone in the red colored regions of the pseudostems colonized by the banana aphid. As recently reported for secondary metabolites of Arabidopsis thaliana and Hypericum species as well as for phenylphenalenone-type secondary metabolites of different € lscher et al., 2009, 2014), LDI-MSI is an effecbanana cultivars (Ho tive technique to identify site-specific secondary metabolites in plant species. LDI-MSI revealed that the location of the four phenylphenalenones 1e4 in the Musa pseudostem was restricted to the red colored regions colonized by P. nigronervosa.
2.3. Laser desorption/ionization mass spectrometric imaging of aphid-colonized pseudostem material of BG
A stereomicroscope Carl Zeiss Stemi SV 11 equipped with a HBO 110 W high pressure mercury plasma arc discharge lamp, an Axio Cam HRc camera, an Axio Vision 4.0 software and a cold light source KL 1500 (Carl Zeiss AG, Oberkochen, Germany) was used to study plant surface areas. 1 H NMR spectra were measured on an Avance 400 NMR
Matrix-free LDI-MSI was used to investigate the presence of phenylphenalenones in the red-colored regions of aphid-colonized pseudostems. The laser beam of the Ultraflex III mass spectrometer
3. Conclusions The occurrence of phenylphenalenones in the red-colored pseudostem and peel material of Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ (BG) suggested that these secondary plant metabolites are phytoalexins involved in the interaction between the banana plant and the banana aphid and the latania scale. However, the rapid exudation of the phenylphenalenones by the banana aphids in the environment and the transfer of these metabolites into its dorsal scales by H. lataniae seem to prevent toxic effects on the insects. 4. Experimental 4.1. General experimental procedures
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
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Fig. 4. Negative ion mode LDI-MSI of red-colored pseudostem material of BG colonized by banana aphids. (A) Optical image of the red regions of BG. (B) The area of the red region subjected to LDI-MSI. (C) and (D) The molecular image of section B for the m/z 271 ([MH]) and m/z 287 ([MH]), respectively, including tissue background (left) and background subtracted (right).
spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at 400.13 MHz. Standard Bruker pulse sequences were used to record spectra in acetone-d6 at 300 K. Spectra were referenced to tetramethylsilane, which was used as an internal standard. For UHPLC, an Ultimate 3000 series RSLC (Dionex, Sunnyvale, CA, USA) was used applying an Acclaim C18 column (150 2.1 mm, 2.2 mm, Dionex, Sunnyvale, CA, USA) with a flow rate of 300 ml min1 in a binary solvent system of water (Solvent A) and acetonitrile (hypergrade for LC MS, Merck, Darmstadt Germany) (Solvent B), both containing 0.1% (v/v) formic acid (eluent additive for LCeMS, Sigma Aldrich, Steinheim, Germany). Sample volumes were loaded onto the column and eluted by using a gradient as follows: linear increase from 0% B to 100% B within 15 mine100% B constant for 5 min e equilibration time at 0% B for 5 min. This system was coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Ionization was accomplished using Electrospray Ionization (ESI). ESI source parameters were set to 4 kV for spray voltage, 35 V for transfer capillary voltage at a capillary temperature 275 C. The samples were measured in positive ion mode in the mass range of m/z 100 to 2000 using 30,000 m/Dm resolving power in the Orbitrap mass analyzer. Data was evaluated and interpreted using XCALIBUR software (Thermo Fisher Scientific Inc., Waltham, MA, USA). An Ultraflex III mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) was used for matrix-free LDI-MSI analysis. The instrument was equipped with a Nd:YAG laser. All spectra were measured in negative reflectron mode.
2015) and subsequently transferred to the Greenhouse for Tropical Crops, University of Kassel in Witzenhausen. Plants were cultivated in pots with 8 l of quartz-peat mixture (2:1) and maintained under greenhouse conditions at an ambient temperature of 27/20 C (day/night), 80% relative humidity, 12 h photoperiod and irrigated as required. Additionally, BG plants were cultivated in custom-made plexiglass containers (diameter: 35 cm, length: 1 m) €tsch Industries GmbH, Typ HPZ in climate chambers (Heraeus Vo 180, Hanau, Germany). The plants were raised in sand, continually irrigated with water, provided a nutrient solution every two weeks and maintained at an ambient temperature of 22 C (day/night), 65% relative humidity.
4.2. Plant material
4.4. Sooty mold
The Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ (Musaceae) was collected in Wadi Tiwi, Oman (Behrendt et al.,
The dark pigmented epifoliar fungi which colonized areas where aphid honeydew was deposited on the surface of the
4.3. Banana aphids All banana cultivars in the greenhouses of the MPI for Chemical Ecology in Jena and of the University of Kassel in Witzenhausen are host plants of the banana aphid Pentalonia nigronervosa Coquerel (Aphididae) and were unintentionally infected with this pest in the greenhouses. The aphid populations are managed in the greenhouse using parasitic wasps (Aphidius ervi Haliday (Braconidae), Aphidius colemani Viereck (Braconidae), Aphidius matricariae Haliday (Braconidae) and Lysiphlebus testaceipes Cresson (Braconidae)) under strict greenhouse biological pest control regulations. After introducing the parasitic wasps, banana aphids in these greenhouses were able to survive primarily between pseudostem and leaf petioles of the banana plants.
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
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petioles of banana varieties in the greenhouse of the University at Kassel in Witzenhausen occurred naturally. The dark mats of sooty molds were collected using soft feeding spoons with rounded edges to avoid damaging epidermal plant tissue. No sooty molds were observed in the greenhouses at the MPI-CE in Jena.
4.7. UHPLC-ESI-MS analysis Sample dilutions of 1:100 were used to analyze phenylphenalenones by injecting 5 ml of the CHCl3 subfractions. In the case of the extract of the dorsal scales of H. lataniae the small amount of phenylphenalenones required a dilution of 1:10 and an injection volume of 7 ml.
4.5. Hemiberlesia lataniae The banana plants in the greenhouse at MPI-CE in Jena were unintentionally infected with the armored scale Hemiberlesia lataniae Signoret (Diaspididae). These insects were not present in the greenhouse at Witzenhausen.
4.6. Isolation and structure elucidation of phenylphenalenones Both green and red colored pseudostem material (86 g fr. wt each) and yellow and red colored exocarp material (86 g fr wt each) of BG plants was manually dissected using scissors, frozen in liquid nitrogen, ground, and exhaustively extracted with ethanol at room temperature. Sooty mold (1.27 g), 250 latania scales and 1000 banana aphids were carefully collected from the plant tissues, vigorously crushed in an Eppendorf tube with plastic pestles for microtubes and exhaustively sonified in ethanol at room temperature. The crude extracts were evaporated (<40 C) and partitioned between trichloromethane-water (CHCl3-H2O), followed by ethyl acetate-water (EtOAc-H2O). The CHCl3 fraction was subjected to PLC (silica gel 60 F254, 20 20 cm, 0.5 mm layer thickness; solvent toluene-acetone (C7H8-Me2CO; 2:1, v:v)). Four colored PLC zones of the CHCl3 subfractions (compound 1: Rf 0.90, compound 2: Rf 0.85, compound 3: Rf 0.64, compound 4: Rf 0.60) were scraped off, eluted with CHCl3, acetone and ethanol, and passed through a RP-18 cartridge (elution with CHCl3, acetone and ethanol). Amounts of compounds isolated from the red-colored pseudostem tissue and from the exocarp of BG were as follows: 1: 4.91/0.39 mg, 2: 2.80/ 0.44 mg, 3: 2.73/2.56 mg, 4: 1.60/1.96 mg. The samples of sooty mold, latania scales and banana aphids were only used for the detection of the four phenylphenalenones 1e4 during the UHPLCESI-MS analysis. Analytical data of anigorufone (1): 1H NMR (acetone-d6, 400.13 MHz) d 8.41 (1H, d, J ¼ 8.2 Hz, H-7), 8.09 (1H, d, J ¼ 8.2 Hz, H6), 7.88 (1H, d, J ¼ 7.1 Hz, H-4), 7.70 (1H, dd, J ¼ 8.2, 7.1 Hz, H-5), 7.63 (1H, d, J ¼ 8.2 Hz, H-8), 7.47e7.38 (5H, m, H-20 -60 ), 7.18 (1H, s, H-3). ESIMS: m/z 273.0912 [MþH]þ, calc. C19H13O2, 273.0910, 0.673 ppm deviation. Analytical data of isoanigorufone (2): 1H NMR (acetone-d6, 400.13 MHz) d 8.72 (1H, d, J ¼ 7.5 Hz, H-9), 8.48 (1H, d, J ¼ 8.1 Hz, H7), 8.14 (1H, d, J ¼ 8.5 Hz, H-6), 7.93 (1H, dd, J ¼ 8.1, 7.5 Hz, H-8), 7.65 (1H, d, J ¼ 8.6 Hz, H-5), 7.61e7.54 (5H, m, H-20 -60 ), 7.14 (1H, s, H-3). ESIMS: m/z 273.0912 [MþH]þ, calc. C19H13O2, 273.0910, 0.673 ppm deviation. Analytical data of hydroxyanigorufone (3): 1H NMR (acetone-d6, 400.13 MHz) d 8.36 (1H, d, J ¼ 8.2 Hz, H-7), 8.05 (1H, d, J ¼ 8.0 Hz, H6), 7.84 (1H, d, J ¼ 7.0 Hz, H-4), 7.66 (1H, dd, J ¼ 8.0, 7.0 Hz, H-5), 7.62 (1H, d, J ¼ 8.2 Hz, H-8), 7.27 (2H, d, J ¼ 8.5 Hz, H-2'/60 ), 7.15 (1H, s, H3), 6.92 (2H, d, J ¼ 8.5 Hz, H-3'/50 ). ESIMS: m/z 289.0861 [MþH]þ, calc. C19H13O3, 273.0910, 0.585 ppm deviation. Analytical data of irenolone (4): 1H NMR (acetone-d6, 400.13 MHz) d 8.71 (1H, d, J ¼ 7.4 Hz, H-9), 8.45 (1H, d, J ¼ 8.1 Hz, H7), 8.10 (1H, d, J ¼ 8.5 Hz, H-6), 7.90 (1H, dd, J ¼ 8.1, 7.4 Hz, H-8), 7.64 (1H, d, J ¼ 8.6 Hz, H-5), 7.40 (2H, d, J ¼ 8.5 Hz, H-2'/60 ), 7.24 (1H, s, H3), 7.08 (2H, d, J ¼ 8.5 Hz, H-3'/50 ). ESIMS: m/z 289.0861 [MþH]þ, calc. C19H13O3, 273.0910, 0.585 ppm deviation.
4.8. Sample preparation and matrix-free LDI-MSI Thin, small parts of the epidermal layer of pseudostem material from BG were manually separated from the rest of the pseudostem and fixed on a carbon-conductive adhesive tape (Plano GmbH, Wetzlar, Germany), which was in turn fixed on an ITO slide (Bruker Daltonik GmbH, Bremen, Germany). A Staedtler triplus gel-liner (silver, 0.4 mm; Staedtler Mars GmbH & Co. KG, Nürnberg, Germany) was used to place marks close to the samples to define their position. The silver gel points served as reference points when overlaying the optical image with the LDI-MSI data. To measure the pixels of approximately 10 10 mm, the minimum laser-focus setting (corresponding to a diameter of about 10 mm laser, under use of oversampling) of the Ultraflex III mass spectrometer and a step size of 10 mm was used. For each raster point, a spectrum was accumulated with 200 laser shots/pixel in the mass range of m/z 60e900 Da and fixed laser intensity. FlexImaging v3.0 software (Bruker Daltonik GmbH, Bremen, Germany) was used to acquire and to process data. Each image corresponding to an m/z value was visualized by overlaying the image with the optical image of the epidermal region. All signal intensities in the m/z image are represented by a specific color corresponding to the mass of the metabolites hereby visualized.
Acknowledgements We wish to thank Dr. Tamara Krügel and the greenhouse team at the MPI-CE (Jena, Germany) and Rainer Braukmann and the team of the Greenhouse for Tropical Crops at the University of Kassel in Witzenhausen for raising the Musa ABB cv. ‘Bluggoe’ plants. We thank Prof. Dr. Stefan Vidal (Department for Crop Sciences, Agri€ ttingen, Germany) and Dr. cultural Entomology, University of Go Thomas Thieme (Bio-Test Labor GmbH Sagerheide, Grob Lüsewitz, Germany) for the identification of the banana aphid, P. nigronervosa. The authors also wish to thank Prof. Dr. Maria Finckh (Ecological Plant Protection, University of Kassel, Germany) for the identification of the dark pigmented epifoliar fungi as sooty molds and Dr. Christoph Hoffmann (Department of Plant Protection in Fruit Crops and Viticulture, Julius-Kühn-Institute Siebeldingen, Germany) for the identifying the armored scales as H. lataniae. We wish to thank Daniel Veit and Frank Müller (MPI-CE) for the construction of the plexiglass containers. The authors gratefully acknowledge Dr. Alexandra zum Felde for editorial help and Dr. Katrin Knop for helpful discussions. This study was supported in part by German Research Foundation (Deutsche Forschungsgemeinschaft (DFG) Grants HO 4380/1 and SCHN 450/10 (to D.H. and B.S.) and the Thüringer Ministerium für Bildung, Wissenschaft und Kultur Grant B515-07008 (to U.S.S.) and the DFG Grant SCHU 1229/15-1 (to U.S.S.) for financial support and instrumentation.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.10.007.
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
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€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
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Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to colonization by Sternorrhyncha € lscher a, b, *, Antje Vollrath c, d, Marco Kai e, 1, Dirk Ho Suganthaguntalam Dhakshinamoorthy f, 2, Riya C. Menezes e, Ales Svatos e, Ulrich S. Schubert c, d, Andreas Buerkert b, Bernd Schneider a, ** €ll-Str. 8, 07745 Jena, Germany Research Group Biosynthesis/NMR, Max Planck Institute for Chemical Ecology, Hans-Kno Organic Plant Production and Agroecosystems Research in the Tropics and Subtropics (OPATS), University of Kassel, Steinstr. 19, 37213 Witzenhausen, Germany c Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena, Humboldtstr. 10, 07743 Jena, Germany d Jena Center of Soft Matter (JCSM), Friedrich Schiller University Jena, Humboldtstr. 10, 07743 Jena, Germany e €ll-Str. 8, 07745 Jena, Germany Research Group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology, Hans-Kno f Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Faculty of Bioscience Engineering, University of Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2016 Received in revised form 10 October 2016 Accepted 12 October 2016 Available online xxx
The interaction of two Sternorrhyncha species, the banana aphid (Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae, Aphidinae)), vector of the banana bunchy top virus (BBTV), and the latania scale (Hemiberlesia lataniae Signoret (Hemiptera: Diaspididae, Diaspidinae)) with Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ (Musaceae) was investigated by a combination of conventional and spatially resolved analytical techniques, 1H NMR, UHPLC-MS, and matrix-free UV-laser desorption/ionization MS imaging. After infestation, the feeding sites of P. nigronervosa on the pseudostem and the exocarp of banana fruit developed a red tinge, in which tissue-specific accumulations of phenylphenalenones were discovered. Phenylphenalenones were also detected in the black mats of sooty molds growing on the banana aphid exudates and in the dorsal scales of H. lataniae. This suggests that although these secondary metabolites play a role in the reaction of banana plants towards attack by sucking insects, an aphid and an armored scale have established mechanisms to exude these metabolites before they deploy their deleterious effect. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Hemiberlesia lataniae Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ Musaceae Pentalonia nigronervosa Aphididae Diaspididae Mass spectrometric imaging Phenylphenalenones Plant-pathogen interaction
1. Introduction Bananas and plantains (Musa spp.) are important food crops in tropical and subtropical areas of the world. In 2013 over 156 million t
* Corresponding author. Organic Plant Production and Agroecosystems Research in the Tropics and Subtropics (OPATS), University of Kassel, Steinstr. 19, 37213 Witzenhausen, Germany. ** Corresponding author. € lscher), [email protected] E-mail addresses: [email protected] (D. Ho (B. Schneider). 1 Present address: Institute of Biological Science, Department of Biochemistry, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany. 2 Present address: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive NW, Atlanta, GA 30332-0100, USA.
bananas and plantains were harvested (FAOSTAT, 2013). These fruits are major staples for more than 400 million people particularly in India, China and many African countries (FAOSTAT, 2013). Fungi, insects, plant-parasitic nematodes and viruses can have serious negative effects on banana yields. The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae) is a known vector of banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus; Fauquet et al., 2005), one of the diseases most heavily affecting banana yields in the Pacific, South and South-East Asia and parts of Africa (Dale, 1987; Kagy et al., 2001; Kumar et al., 2011). P. nigronervosa was first described in the 19th century on the union (Coquerel, 1859), although the aphid likely origisland of Re inated in Southeast Asia, as did the genus Musa (Waterhouse, 1987; Robinson, 1996). P. nigronervosa is now widely distributed in the
http://dx.doi.org/10.1016/j.phytochem.2016.10.007 0031-9422/© 2016 Elsevier Ltd. All rights reserved.
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
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€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
tropics and in greenhouses in Europe and North America (Blackman and Eastop, 1984) but appears to specifically affect plants of the genus Musa (Foottit et al., 2010). BBTV is the causal agent of banana bunchy top disease (BBTD) and the banana aphid transmits the virus in a circulative, nonpropagative manner (Magee, 1927; Hu et al., 1996). While the Sigatoka disease complex (caused by Mycosphaerella spp. Johanson, Mycosphaerellaceae) may cause yield losses of up to 30e50% (Mobambo et al., 1993), plants infected gi et al., 1995). with BBTV may become completely unproductive (Sa The movement of infected material is thought to spread the virus over long distances and, once established, the disease is difficult to manage and eradicate (Banerjee et al., 2014). Recent publications addressing the interaction between the banana aphid, BBTV and banana plants have focused on strategies for controlling BBTV (Robson et al., 2006; Hooks et al., 2009) and the development of transgenic banana germplasm to manage the virus (Elayabalan et al., 2013). BBTV is known to infect a range of Musa species, including M. coccinea Andrews (Musaceae), M. ornata Roxb. (Musaceae), M. balbisiana Colla (Musaceae) and M. acuminata ssp. zebrina (Van Houtte ex Planch) Nasution (Musaceae) (Ploetz et al., 2015). In our greenhouses, banana aphid colonies were discovered on red regions of the pseudostem of Musa acuminata balbisiana Colla (ABB group) ‘Bluggoe’ (Musaceae) (BG). BG is a popular starchy banana primarily used for cooking, is grown in many countries and recognized to be resistant to drought and Black and Yellow Sigatoka disease but susceptible to Panama disease (Jones, 1999). In the greenhouse of the University of Kassel in Witzenhausen (Germany), some areas between pseudostem and the base of leaf petioles were colonized by dark pigmented epifoliar fungi (sooty molds) which are reported to take no nutrients or water from the host plants (Hughes, 1976), are not known to induce phytoalexin responses by host plants, but can negatively affect plant growth by reducing the rate of photosynthesis. In the greenhouse of the Max Planck Institute for Chemical Ecology (MPI-CE) in Jena (Germany), an additional sucking insect, latania scale (Hemiberlesia lataniae Signoret, Diaspididae) was observed to colonize Musa spp. H. lataniae belongs to the family of armored scales (Diaspididae), a group of plant parasitic arthropods including some of the most damaging pests of perennial crops and ornamentals (Beardsley and Gonzalez, 1975; Miller et al., 2005). The latania scale is highly polyphagous and occurs throughout the world in tropical to temperate zones (Miller and Davidson, 2005; Normark and Johnson, 2011; CABI, 2015). In the case of bananas, some species of armored scales (Hemiberlesia musae Takagi & Yamamoto (Diaspididae) and Hemiberlesia ocellata Takagi & Yamamoto (Diaspididae)) have been observed on infested imported banana fruits (Takagi and Yamamoto, 1974). The coconut scale, Aspidiotus destructor Signoret (Diaspididae), was reported to cause heavy infestations resulting in yellowing and necrosis of leaves of banana in Hawaii (Wright and Diez, 2005). Many plant secondary metabolites possess insecticidal activities. Deterrent effects of plant metabolites such as the isoflavone genistein and the flavone luteolin on phloem-sucking insects such as the pea aphid (Acyrthosiphon pisum Harris (Aphididae)) prompted breeding of plants resistant to phloem-feeding insects (Goławska and Łukasik, 2012). Phenylphenalenones play a major € lscher et al., 2014). In role in the banana plant defense system (Ho Musa spp., the formation of phenylphenalenone-related compounds has been elicited in the peels of fruit by Colletotrichum musae (Berk. & M.A. Curtis) Arx (Glomerellaceae) (Anthracnose disease), in leaves by Mycosphaerella fijiensis Morelet (Mycosphaerellaceae) (Black Sigatoka leaf streak disease), and in roots and rhizomes by Fusarium oxysporum f. sp. cubense (E.F.Sm.) W.C.Snyder & H.N.Hansen (Nectriaceae) (Panama disease) (Luis et al., 1994; Ot alvaro et al., 2007; Hirai et al., 1994). A combination of
conventional and spatially resolved analytical techniques (NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging (LDI-MSI), and Raman microspectroscopy) was applied to identify and locate phenylphenalenones in lesions caused by the burrowing nematode Radopholus similis (Cobb) Thorne (Pratylenchidae) in the roots of banana cultivars differing in €lscher et al., 2014). The study of this susceptibility to R. similis (Ho interaction provided evidence for the local induction of phenylphenalenone-type secondary metabolites in response to R. similis infection in Musa spp. The banana-nematode interaction demonstrated the effects of phenylphenalenone-type compounds on an important pathogen of banana plants. The present study reports the phenylphenalenones occurring in the red regions of the pseudostem and the fruit exocarp of BG that were colonized by P. nigronervosa, in the black mats of dark pigmented epifoliar fungi (sooty molds) and the dorsal scales of H. lataniae. 2. Results and discussion 2.1. Pseudostem and banana peel discoloration e banana aphids, sooty molds and armored scales In the greenhouse, BG plants were colonized with P. nigronervosa (Fig. 1A) between pseudostem and petiole leaf and on banana fruits. The pseudostems of one year old plants and four week old banana fruits were monitored for aphid damage. The development of a red tinge was observed on the surface of aphidcolonized plant tissues (Fig. 1BeE), while aerial, aphid free areas of the banana plants appeared healthy upon visual inspection. As leaf petioles grew away from the pseudostem, the red-colored areas which were also covered by aphid honeydew were exposed to epifoliar fungi and sooty mold mats developed on them (Fig. 1F). In the MPI-CE greenhouse, banana plants were additionally colonized by armored scales (H. lataniae). The latania scales caused dark red regions on the banana pseudostems (Fig. 1G and H). It appears that the observed coloration is the response of banana plants to attack by the banana aphid and the armored scale, respectively. Armored scale infestations have been reported to cause leaf chlorosis and the development of colored spots on different plant species such as coconuts, other palms and pineapples (Beardsley and Gonzalez, 1975). Olive scales (Parlatoria oleae Colvee (Diaspididae)) induce purple spots on the green olives turning straw colored as the fruits mature (McKenzie, 1952). Recently Hill et al. (2011) showed the development of bowl shaped wound periderm encircling latania scales on the canes of some genotypes of kiwifruit (Actinidia chinensis L. (Actinidiaceae)). They report that an extensive deposition of phenolic compounds within the suberized/lignified wound periderm cells and cortical cells beneath and around the insect prevents the scales stylet from reaching unmodified parenchyma tissue on which this diaspidid scale could feed, ultimately leading to the death of the scale. As opposed to the lethal response of some kiwifruit genotypes on latania scales on the canes, the formation of a red tinge on the pseudostem and the fruit exocarp of BG did not kill the banana aphid or the latania scale. Nonetheless, the eurymerous behavior of latania scales allows the survival of members of this species on kiwifruit plants. 2.2. Isolation, identification and function of plant secondary metabolites The red colored areas of the pseudostem from BG and the fruit exocarp of BG were manually dissected using scissors. Green (pseudostem) and yellow (exocarp) healthy control material was additionally checked under a stereo microscope for the appearance
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
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Fig. 1. Photos of sucking insects and their damage on BG tissues. (A) An adult banana aphid (P. nigronervosa) on red-colored pseudostem material of BG. (B, C) Dark red colored regions of the pseudostem of BG colonized by the banana aphid. (D) Red colored areas on the peel of a ripe and a green BG banana fruit, covered with the shed skins of banana aphids (E). (F) Black mats of sooty mold on BG pseudostem. (G) Adult H. lataniae on red colored areas of BG pseudostems and (H) a single female adult latania scale with its membranous ventral scale separating the insect from direct contact with the plant surface and the experimentally removed dorsal scale on the right side of the photo.
of putative small red-colored regions, which were removed with a scalpel and discarded. The mesocarp of the banana fruit was removed from the exocarp using a spoon with rounded edges. The dark pigmented epifoliar sooty molds were removed from the surface of the pseudostem in the same way. A microspoon with rounded edges was used to collect 500 mg dorsal scales of H. lataniae. All samples were separately subjected to extraction using ethanol. Liquid-liquid separation resulted in CHCl3, ethyl acetate and aqueous subfractions. 1H NMR analysis of the CHCl3 subfraction of the P. nigronervosa-colonized red pseudostem material showed signals in the aromatic range of the spectrum while the corresponding fraction obtained from green tissue did not show such signals. The CHCl3 subfraction of the red tissue was then purified by preparative layer chromatography (PLC). The purified compounds were subjected to 1H NMR spectroscopy and four compounds were identified as anigorufone (1), isoanigorufone (2), hydroxyanigorufone (3), and irenolone (4) (Fig. 2; Cooke and Thomas, 1975; Luis et al., 1993, 1999). All the compounds are known as typical metabolites and phytoalexins of Musa species lvaro et al., 2007). (Luis et al., 1994; Hirai et al., 1994; Ota Equal amounts of all the CHCl3-subfractions were subjected to UHPLC-ESI-MS analysis (Fig. 3), with the exception of the CHCl3subfraction of the dorsal scales of H. lataniae. The small levels of phenylphenalenones occurring in the latter sample required the
OH R
O
1R=H 3 R = OH
OH R
O
2R=H 4 R = OH
Fig. 2. Structures of isolated phenylphenalenone-type phytoalexins 1e4. (1) Anigorufone, (2) isoanigorufone, (3) hydroxyanigorufone and (4) irenolone.
Fig. 3. Extracted-ion chromatograms of a UHPLC-ESI-MS analyses of CHCl3-subfractions of ethanolic extracts obtained from red areas of the P. nigronervosa colonized BG pseudostem (red line), from red regions from P. nigronervosa-colonized BG exocarp (blue line), from sooty mold colonizing P. nigronervosa honeydew (brown line), from the dorsal scales of H. lataniae (pink line) and from adults of P. nigronervosa (green line). (1) Anigorufone, (2) isoanigorufone, (3) hydroxyanigorufone and (4) irenolone.
use of a less diluted sample (1:10 instead of 1:100). The phytochemical profiles of the red regions of the BG pseudostem, the dorsal scales of H. lataniae and the sooty mold showed a high similarity concerning the occurrence of the four phenylphenalenones 1e4. A better resolution for the result of the dorsal scales of H. lataniae is shown in Fig. S1 (Supporting information). The amount of anigorufone (1) and isoanigorufone (2) is higher than the amount of the two p-hydroxylated phenylphenalenones (3 and 4). In the red regions of the BG fruit exocarp, hydroxyanigorufone (3) and irenolone (4) are the major compounds in comparison with anigorufone (1) and isoanigorufone (2). These results are
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€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
an excellent example of the capability of different parts of banana plants to respond with different phytochemical profiles to a pest. The spectra of the banana aphid extracts (Fig. 3) as the spectra of the controls showed no evidence of the compounds 1e4. The banana aphids take up substantial amounts of host plant content (Leroy et al., 2011) to extract nitrogen-containing nutrients, constantly exuding honeydew, and presumably not allowing phenylphenalenones to accumulate in their bodies. The proof of phenylphenalenones in the sooty molds was the first hint that phenylphenalenones are transported through the bodies of aphids and, without being markedly metabolized, are exuded into their surroundings. Under the greenhouse conditions at the University of Kassel in Witzenhausen, aphids colonized the area below the Vshape formed by leaf petioles and pseudostems of banana plants. Due to this location, their exudates accumulated on the inner side of the leaf petiole over time. Sooty molds colonized the exudates of aphid colonies which had accumulated over the period of one year. The material which was investigated therefore contained nonvolatile secondary metabolites exuded by the banana aphids over the period of complete leaf expansion (Fig. 3). In contrast, banana aphid extracts were collected from individuals with a maximum lifespan of four weeks, during which time it is presumed that they continuously exude honeydew containing the plant secondary metabolites. The absence of phenylphenalenones in the aphid's body extract is strikingly different from findings of a recent study of different Musa-derived phenylphenalenone-type compounds in the burrowing nematode R. similis. Anigorufone (1), which caused the greatest nematostatic and nematocidal effects among the phenylphenalenones tested, accumulated in the body of the nem€ lscher et al., 2014). Further evidence for a lack of detriatode (Ho mental effects of phenylphenalenones on P. nigronervosa was obtained when we cultivated different bananas varieties in plexiglass containers in the presence of banana aphids; none of the plants in infested containers survived the attack. Obviously phenylphenalenones are ineffective agents against banana aphids because the metabolites passed through these insects without apparently harming them. Thus there was no reason to assay deterrent or toxic effects of phenylphenalenones on the aphids. Different from P. nigronervosa, the exudates of H. lataniae are not distributed within the neighboring space but stored in their dorsal scales. Armored scale insects do not excrete honeydew and lack the filter chamber type of digestive system found in most other Coccoidea (Beardsley and Gonzalez, 1975). It has been shown that the material excreted through the anus is used in the formation of the dorsal scale (Disselkamp, 1954; Beardsley and Gonzalez, 1975). The presence of phenylphenalenones in these dorsal scales indicates that H. lataniae also takes up and exudes these plant secondary metabolites without being noticeably affected by them. Transcriptome analysis of kiwifruit bark in response to latania scales and of banana plants to C. musae, respectively, showed the significant upregulation of transcripts associated with secondary metabolism (Tang et al., 2013; Hill et al., 2015). The occurrence of transcripts specific for enzyme orthologues including phenylalanine ammonia lyase, chalcone synthase and coumarate CoA ligase which can be involved in the biosynthesis of either phenylphenalenones or phenolic compounds (i.e. flavonoids and tannins) can be regarded as further evidence for the role of these secondary metabolites in the pathogen defense response of plants.
can be focused on spots down to 10 mm to allow spatial information of phenylphenalenones in the red-colored pseudostem regions. It revealed the accumulation of secondary metabolites in the aphidcolonized regions. The LDI-MSI images were plotted in FlexImaging software 3.0 after spectra baseline correction, hotspot removal (by suppressing 1% of the most intense pixels), and edge-preserving image-denoising to reduce the pixel-to-pixel variation. The m/zvalues of the discovered plant secondary metabolites in the red pseudostem region are consistent with the four phenylphenalenones (Fig. 2) from the phytochemical investigation of the aphid-infested material. A signal for anigorufone (1) and isoanigorufone (2) (m/z 271, [MH]) was found in this area and the m/z 287 ([MH]) value detected within the red colored pseudostem region was assigned to [MH] of hydroxyanigorufone (3) and irenolone (4) (Fig. 4). The green aerial parts and the red pseudostem regions colonized by the banana aphids were strikingly different from each other with regard to the presence of phenylphenalenones, both in the phytochemical extractions and identifications as well as in the LDI-MSI investigations. The formation of phenylphenalenone-type compounds by bananas has been reported as a response to infection with R. similis (burrowing nematode), C. musae (Anthracnose), M. fijiensis (Black Sigatoka) and F. oxysporum f. sp. cubense (Panama disease; Luis lvaro et al., 2007; Hirai et al., 1994; Ho €lscher et al., et al., 1994; Ota 2014; Hidalgo et al., 2016). The present phytochemical investigations provide evidence for the local induction of phenylphenalenones in Musa ABB cv. ‘Bluggoe’ in response to the banana aphid and the latania scale and extend the number of banana pathogens that elicit the site-specific formation of phenylphenalenone-related compounds. Four typical phytoalexins of the phenylphenalenone-type were shown by NMR and LDI-MSI analyses to be present in the affected pseudostem tissues of Musa plants. Anigorufone (1) was the most abundant phenylphenalenone in the red colored regions of the pseudostems colonized by the banana aphid. As recently reported for secondary metabolites of Arabidopsis thaliana and Hypericum species as well as for phenylphenalenone-type secondary metabolites of different € lscher et al., 2009, 2014), LDI-MSI is an effecbanana cultivars (Ho tive technique to identify site-specific secondary metabolites in plant species. LDI-MSI revealed that the location of the four phenylphenalenones 1e4 in the Musa pseudostem was restricted to the red colored regions colonized by P. nigronervosa.
2.3. Laser desorption/ionization mass spectrometric imaging of aphid-colonized pseudostem material of BG
A stereomicroscope Carl Zeiss Stemi SV 11 equipped with a HBO 110 W high pressure mercury plasma arc discharge lamp, an Axio Cam HRc camera, an Axio Vision 4.0 software and a cold light source KL 1500 (Carl Zeiss AG, Oberkochen, Germany) was used to study plant surface areas. 1 H NMR spectra were measured on an Avance 400 NMR
Matrix-free LDI-MSI was used to investigate the presence of phenylphenalenones in the red-colored regions of aphid-colonized pseudostems. The laser beam of the Ultraflex III mass spectrometer
3. Conclusions The occurrence of phenylphenalenones in the red-colored pseudostem and peel material of Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ (BG) suggested that these secondary plant metabolites are phytoalexins involved in the interaction between the banana plant and the banana aphid and the latania scale. However, the rapid exudation of the phenylphenalenones by the banana aphids in the environment and the transfer of these metabolites into its dorsal scales by H. lataniae seem to prevent toxic effects on the insects. 4. Experimental 4.1. General experimental procedures
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Fig. 4. Negative ion mode LDI-MSI of red-colored pseudostem material of BG colonized by banana aphids. (A) Optical image of the red regions of BG. (B) The area of the red region subjected to LDI-MSI. (C) and (D) The molecular image of section B for the m/z 271 ([MH]) and m/z 287 ([MH]), respectively, including tissue background (left) and background subtracted (right).
spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) at 400.13 MHz. Standard Bruker pulse sequences were used to record spectra in acetone-d6 at 300 K. Spectra were referenced to tetramethylsilane, which was used as an internal standard. For UHPLC, an Ultimate 3000 series RSLC (Dionex, Sunnyvale, CA, USA) was used applying an Acclaim C18 column (150 2.1 mm, 2.2 mm, Dionex, Sunnyvale, CA, USA) with a flow rate of 300 ml min1 in a binary solvent system of water (Solvent A) and acetonitrile (hypergrade for LC MS, Merck, Darmstadt Germany) (Solvent B), both containing 0.1% (v/v) formic acid (eluent additive for LCeMS, Sigma Aldrich, Steinheim, Germany). Sample volumes were loaded onto the column and eluted by using a gradient as follows: linear increase from 0% B to 100% B within 15 mine100% B constant for 5 min e equilibration time at 0% B for 5 min. This system was coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Ionization was accomplished using Electrospray Ionization (ESI). ESI source parameters were set to 4 kV for spray voltage, 35 V for transfer capillary voltage at a capillary temperature 275 C. The samples were measured in positive ion mode in the mass range of m/z 100 to 2000 using 30,000 m/Dm resolving power in the Orbitrap mass analyzer. Data was evaluated and interpreted using XCALIBUR software (Thermo Fisher Scientific Inc., Waltham, MA, USA). An Ultraflex III mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) was used for matrix-free LDI-MSI analysis. The instrument was equipped with a Nd:YAG laser. All spectra were measured in negative reflectron mode.
2015) and subsequently transferred to the Greenhouse for Tropical Crops, University of Kassel in Witzenhausen. Plants were cultivated in pots with 8 l of quartz-peat mixture (2:1) and maintained under greenhouse conditions at an ambient temperature of 27/20 C (day/night), 80% relative humidity, 12 h photoperiod and irrigated as required. Additionally, BG plants were cultivated in custom-made plexiglass containers (diameter: 35 cm, length: 1 m) €tsch Industries GmbH, Typ HPZ in climate chambers (Heraeus Vo 180, Hanau, Germany). The plants were raised in sand, continually irrigated with water, provided a nutrient solution every two weeks and maintained at an ambient temperature of 22 C (day/night), 65% relative humidity.
4.2. Plant material
4.4. Sooty mold
The Musa acuminata balbisiana Colla (ABB Group) ‘Bluggoe’ (Musaceae) was collected in Wadi Tiwi, Oman (Behrendt et al.,
The dark pigmented epifoliar fungi which colonized areas where aphid honeydew was deposited on the surface of the
4.3. Banana aphids All banana cultivars in the greenhouses of the MPI for Chemical Ecology in Jena and of the University of Kassel in Witzenhausen are host plants of the banana aphid Pentalonia nigronervosa Coquerel (Aphididae) and were unintentionally infected with this pest in the greenhouses. The aphid populations are managed in the greenhouse using parasitic wasps (Aphidius ervi Haliday (Braconidae), Aphidius colemani Viereck (Braconidae), Aphidius matricariae Haliday (Braconidae) and Lysiphlebus testaceipes Cresson (Braconidae)) under strict greenhouse biological pest control regulations. After introducing the parasitic wasps, banana aphids in these greenhouses were able to survive primarily between pseudostem and leaf petioles of the banana plants.
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petioles of banana varieties in the greenhouse of the University at Kassel in Witzenhausen occurred naturally. The dark mats of sooty molds were collected using soft feeding spoons with rounded edges to avoid damaging epidermal plant tissue. No sooty molds were observed in the greenhouses at the MPI-CE in Jena.
4.7. UHPLC-ESI-MS analysis Sample dilutions of 1:100 were used to analyze phenylphenalenones by injecting 5 ml of the CHCl3 subfractions. In the case of the extract of the dorsal scales of H. lataniae the small amount of phenylphenalenones required a dilution of 1:10 and an injection volume of 7 ml.
4.5. Hemiberlesia lataniae The banana plants in the greenhouse at MPI-CE in Jena were unintentionally infected with the armored scale Hemiberlesia lataniae Signoret (Diaspididae). These insects were not present in the greenhouse at Witzenhausen.
4.6. Isolation and structure elucidation of phenylphenalenones Both green and red colored pseudostem material (86 g fr. wt each) and yellow and red colored exocarp material (86 g fr wt each) of BG plants was manually dissected using scissors, frozen in liquid nitrogen, ground, and exhaustively extracted with ethanol at room temperature. Sooty mold (1.27 g), 250 latania scales and 1000 banana aphids were carefully collected from the plant tissues, vigorously crushed in an Eppendorf tube with plastic pestles for microtubes and exhaustively sonified in ethanol at room temperature. The crude extracts were evaporated (<40 C) and partitioned between trichloromethane-water (CHCl3-H2O), followed by ethyl acetate-water (EtOAc-H2O). The CHCl3 fraction was subjected to PLC (silica gel 60 F254, 20 20 cm, 0.5 mm layer thickness; solvent toluene-acetone (C7H8-Me2CO; 2:1, v:v)). Four colored PLC zones of the CHCl3 subfractions (compound 1: Rf 0.90, compound 2: Rf 0.85, compound 3: Rf 0.64, compound 4: Rf 0.60) were scraped off, eluted with CHCl3, acetone and ethanol, and passed through a RP-18 cartridge (elution with CHCl3, acetone and ethanol). Amounts of compounds isolated from the red-colored pseudostem tissue and from the exocarp of BG were as follows: 1: 4.91/0.39 mg, 2: 2.80/ 0.44 mg, 3: 2.73/2.56 mg, 4: 1.60/1.96 mg. The samples of sooty mold, latania scales and banana aphids were only used for the detection of the four phenylphenalenones 1e4 during the UHPLCESI-MS analysis. Analytical data of anigorufone (1): 1H NMR (acetone-d6, 400.13 MHz) d 8.41 (1H, d, J ¼ 8.2 Hz, H-7), 8.09 (1H, d, J ¼ 8.2 Hz, H6), 7.88 (1H, d, J ¼ 7.1 Hz, H-4), 7.70 (1H, dd, J ¼ 8.2, 7.1 Hz, H-5), 7.63 (1H, d, J ¼ 8.2 Hz, H-8), 7.47e7.38 (5H, m, H-20 -60 ), 7.18 (1H, s, H-3). ESIMS: m/z 273.0912 [MþH]þ, calc. C19H13O2, 273.0910, 0.673 ppm deviation. Analytical data of isoanigorufone (2): 1H NMR (acetone-d6, 400.13 MHz) d 8.72 (1H, d, J ¼ 7.5 Hz, H-9), 8.48 (1H, d, J ¼ 8.1 Hz, H7), 8.14 (1H, d, J ¼ 8.5 Hz, H-6), 7.93 (1H, dd, J ¼ 8.1, 7.5 Hz, H-8), 7.65 (1H, d, J ¼ 8.6 Hz, H-5), 7.61e7.54 (5H, m, H-20 -60 ), 7.14 (1H, s, H-3). ESIMS: m/z 273.0912 [MþH]þ, calc. C19H13O2, 273.0910, 0.673 ppm deviation. Analytical data of hydroxyanigorufone (3): 1H NMR (acetone-d6, 400.13 MHz) d 8.36 (1H, d, J ¼ 8.2 Hz, H-7), 8.05 (1H, d, J ¼ 8.0 Hz, H6), 7.84 (1H, d, J ¼ 7.0 Hz, H-4), 7.66 (1H, dd, J ¼ 8.0, 7.0 Hz, H-5), 7.62 (1H, d, J ¼ 8.2 Hz, H-8), 7.27 (2H, d, J ¼ 8.5 Hz, H-2'/60 ), 7.15 (1H, s, H3), 6.92 (2H, d, J ¼ 8.5 Hz, H-3'/50 ). ESIMS: m/z 289.0861 [MþH]þ, calc. C19H13O3, 273.0910, 0.585 ppm deviation. Analytical data of irenolone (4): 1H NMR (acetone-d6, 400.13 MHz) d 8.71 (1H, d, J ¼ 7.4 Hz, H-9), 8.45 (1H, d, J ¼ 8.1 Hz, H7), 8.10 (1H, d, J ¼ 8.5 Hz, H-6), 7.90 (1H, dd, J ¼ 8.1, 7.4 Hz, H-8), 7.64 (1H, d, J ¼ 8.6 Hz, H-5), 7.40 (2H, d, J ¼ 8.5 Hz, H-2'/60 ), 7.24 (1H, s, H3), 7.08 (2H, d, J ¼ 8.5 Hz, H-3'/50 ). ESIMS: m/z 289.0861 [MþH]þ, calc. C19H13O3, 273.0910, 0.585 ppm deviation.
4.8. Sample preparation and matrix-free LDI-MSI Thin, small parts of the epidermal layer of pseudostem material from BG were manually separated from the rest of the pseudostem and fixed on a carbon-conductive adhesive tape (Plano GmbH, Wetzlar, Germany), which was in turn fixed on an ITO slide (Bruker Daltonik GmbH, Bremen, Germany). A Staedtler triplus gel-liner (silver, 0.4 mm; Staedtler Mars GmbH & Co. KG, Nürnberg, Germany) was used to place marks close to the samples to define their position. The silver gel points served as reference points when overlaying the optical image with the LDI-MSI data. To measure the pixels of approximately 10 10 mm, the minimum laser-focus setting (corresponding to a diameter of about 10 mm laser, under use of oversampling) of the Ultraflex III mass spectrometer and a step size of 10 mm was used. For each raster point, a spectrum was accumulated with 200 laser shots/pixel in the mass range of m/z 60e900 Da and fixed laser intensity. FlexImaging v3.0 software (Bruker Daltonik GmbH, Bremen, Germany) was used to acquire and to process data. Each image corresponding to an m/z value was visualized by overlaying the image with the optical image of the epidermal region. All signal intensities in the m/z image are represented by a specific color corresponding to the mass of the metabolites hereby visualized.
Acknowledgements We wish to thank Dr. Tamara Krügel and the greenhouse team at the MPI-CE (Jena, Germany) and Rainer Braukmann and the team of the Greenhouse for Tropical Crops at the University of Kassel in Witzenhausen for raising the Musa ABB cv. ‘Bluggoe’ plants. We thank Prof. Dr. Stefan Vidal (Department for Crop Sciences, Agri€ ttingen, Germany) and Dr. cultural Entomology, University of Go Thomas Thieme (Bio-Test Labor GmbH Sagerheide, Grob Lüsewitz, Germany) for the identification of the banana aphid, P. nigronervosa. The authors also wish to thank Prof. Dr. Maria Finckh (Ecological Plant Protection, University of Kassel, Germany) for the identification of the dark pigmented epifoliar fungi as sooty molds and Dr. Christoph Hoffmann (Department of Plant Protection in Fruit Crops and Viticulture, Julius-Kühn-Institute Siebeldingen, Germany) for the identifying the armored scales as H. lataniae. We wish to thank Daniel Veit and Frank Müller (MPI-CE) for the construction of the plexiglass containers. The authors gratefully acknowledge Dr. Alexandra zum Felde for editorial help and Dr. Katrin Knop for helpful discussions. This study was supported in part by German Research Foundation (Deutsche Forschungsgemeinschaft (DFG) Grants HO 4380/1 and SCHN 450/10 (to D.H. and B.S.) and the Thüringer Ministerium für Bildung, Wissenschaft und Kultur Grant B515-07008 (to U.S.S.) and the DFG Grant SCHU 1229/15-1 (to U.S.S.) for financial support and instrumentation.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.10.007.
€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007
€lscher et al. / Phytochemistry xxx (2016) 1e7 D. Ho
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€lscher, D., et al., Local phytochemical response of Musa acuminata balbisiana Colla cv. ‘Bluggoe’ (ABB) to Please cite this article in press as: Ho colonization by Sternorrhyncha, Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.10.007