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Toxic Ipomeamarone Accumulation in Healthy Parts of Sweetpotato (Ipomoea batatas L. Lam) Storage Roots on Infection by Rhizopus stolonifer Lydia Nanjala Wamalwa, Xavier Cheseto, Elizabeth Ouna, Fatma Kaplan, Nguya Maniania, Jesse Machuka, Baldwyn Torto, and Marc Ghislain J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Nov 2014 Downloaded from http://pubs.acs.org on November 27, 2014

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Journal of Agricultural and Food Chemistry

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Toxic Ipomeamarone Accumulation in Healthy Parts of Sweetpotato (Ipomoea batatas L.

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Lam) Storage Roots on Infection by Rhizopus stolonifer

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Lydia N. Wamalwa†,#; Xavier Cheseto‡; Elizabeth Ouna‡; Fatma Kaplan§,ζ; Nguya K.

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Maniania‡; Jesse Machuka#; Baldwyn Torto‡; Marc Ghislain*†

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†

International Potato Centre, P.O. Box 25171-00603 Nairobi, Kenya

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‡

International Centre of Insect Physiology and Ecology (ICIPE) - African Insect Science for

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Food and Health P.O. Box 30772-00100 Nairobi, Kenya

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§

Department of Biology, University of Florida, Gainesville, Florida, USA

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#

Kenyatta University, P.O. Box 43844-00100 Nairobi, Kenya

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ζ

Kaplan Schiller Research, LLC., Gainesville, Florida, USA

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*Corresponding author

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Phone: 254 (020) 4223641; Fax: 254 (020 4223600); Email: [email protected]

16 17

1

ACS Paragon Plus Environment


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Abstract

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Furanoterpenoid accumulation in response to microbial attack in rotting sweetpotatoes has long

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been linked to deaths and lung edema of cattle in the world. However, it is not known whether

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furanoterpenoid, ipomeamarone accumulates in the healthy-looking parts of infected sweetpotato

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storage roots. This is critical for effective utilization as animal feed and assessing the potential

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negative impact on human health. Therefore, we first identified the fungus from infected

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sweetpotatoes as MI-1 (Rhizopus stolonifer) and then infected healthy sweetpotato samples to

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determine furanoterpenoid content. Ipomeamarone and its precursor, dehydroipomeamarone,

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were identified through spectroscopic analyses and its concentration was at toxic levels in

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healthy-looking regions. Dehydroipomeamarone and ipomeamarone were detected in all the

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samples and controls at varying concentrations. Our study provides fundamental information on

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furanoterpenoids in relation to high levels reported that could subsequently affect cattle on

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consumption and high ipomeamarone levels in healthy-looking tissues.

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Keywords: Sweetpotato, furanoterpenoids, ipomeamarone, animal and human health safety,

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Rhizopus stolonifer

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Introduction

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Despite the prevalence of respiratory infections and deaths relating to furanoterpenoids on global

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health of cattle and possibly humans, the concentration levels of these compounds in diseased

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and healthy-looking tissues of infected sweetpotatoes is poorly understood and insufficiently

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documented. Phytoalexins are inducible metabolites elicited by biotic and abiotic factors.

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Sweetpotato elicits several phytoalexins, collectively known as furanoterpenoids including

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ipomeamarone, 1, dehydroipomeamarone, 2, 4-ipomeanol, 3, and 1,4-ipomeadiol, 4 (Figure 1).1,

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2

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cause hepatoxicity, pneumonia, lung edema

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furanoterpenoids is available on isolation methods 7 and its presence in sweetpotatoes 2, 8 there is

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no documented or accessible information on variation of the concentrations from inoculation

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point on towards healthy-looking tissues of an infected sample. This is important because in sub-

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Saharan Africa (SSA), the sweetpotato is a food crop for many rural poor families whereby both

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healthy and infected sweetpotato storage roots are harvested together. The infected parts are

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usually removed and fed to farm animals while the remaining apparently healthy parts are

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consumed by the farm households, typically in western Kenya (Dr. Robert Mwanga, sweetpotato

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breeder, personal communication).

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Sweetpotato is ranked the third most important root and tuber crop after potato and cassava in

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SSA. As a food crop, sweetpotato is used by many poor families due to the higher yield in dry

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matter content per unit area compared to cereal cultivation on an equivalent piece of land.9 In

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addition, sweetpotatoes are particularly important during dry periods of the year when cereal

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crops are unavailable.9 Unfortunately, it is the same dry period that the crop is attacked by

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insects and microbes.10, 11 It is also known that farmers in SSA practice piece-meal harvesting,

Biotic factors such as fungi are reported to elicit varying levels of furanoterpenoids,2 which 3-5

and cattle deaths.6 Although information on

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which exacerbates the storage roots left in the soil during dry seasons to exposure to insects and

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microbes.12 Farmers who harvest sweetpotato storage roots for sale are faced with post-harvest

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infection. At post-harvest, microbes attack the storage roots owing to the favorable warm

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conditions and bruising caused by handling.13 Storage roots respond to attack by eliciting

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production of furanoterpenoids to fight off the infection within the sweetpotato plant. The total

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furanoterpenoid level range of three compounds was documented between 25-67,000 mg/kg in

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harvested storage root samples irrespective of the microbe that elicited this.1,

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radioactivity to analyze ipomeamarone levels has been done but more sensitive techniques such

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as coupled liquid chromatography-quadruple time of flight mass spectrometry (LC-QToF-MS)

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and coupled gas chromatography-mass spectrometry (GC-MS) are needed.14

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It is not known how far into the healthy-looking storage root samples that these furanoterpenoids

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are found and at what levels. Since this has a potential to expose both humans and farm animals

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to either chronic or acute toxicity, we (i) isolated and identified a fungus from the field samples

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that elicited furanoterpenoid production; (ii) characterized the furanoterpenoids using GC-MS

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and LC-Qtof-MS; (iii) evaluated the furanoterpenoid concentration levels produced for different

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sweetpotato cultivars after infection by the isolated fungus; and (iv) determined the

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furanoterpenoid levels within the apparently healthy part of an infected storage root.

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Materials and Methods

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Plant Materials

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Two types of sweetpotato (Ipomoea batatas L. Lam.) samples were used to analyze for

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ipomeamarone concentrations: weevil-infected sweetpotatoes for fungal isolation and healthy

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sweetpotatoes for inoculation experiments with the isolated fungus. Healthy sweetpotatoes were

2

The use of

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purchased from markets in Nairobi, Kenya and were classified by the vendors as Kemb, Naspot,

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Bungoma and Nyawo cultivars. Weevil-infested sweetpotato storage roots were sampled from a

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farm at the Kenya Agricultural Research Institute (KARI), Marigat, Kenya, during the dry season

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when infestation was high. Sampled storage roots were carefully placed in covered but aerated

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plastic jars (20.5 x 10.5 cm) immediately after harvest and then incubated at 25-28 °C for 5 d to

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promote growth of microbes.

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Isolation and identification of Fungus from Weevil-infested Sweetpotatoes

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Isolation was carried out from fully-infected storage roots in jars by using a pin and plated on

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potato dextrose agar (PDA) (Oxoid, Hampshire, England) media supplemented with 50 µg/mL

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streptomycin sulfate (Sigma, St. Louis, MO) and chloramphenicol (25 µg/mL) (Sigma, St. Louis,

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MO) to inhibit bacterial growth. Plates were incubated overnight at 25 °C during which several

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colonies emerged. Single colonies were isolated and sub-cultured on PDA without antibiotics.

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There was a colony that was consistent on all the PDA plates and was named Marigat isolate-1

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(MI-1). It was identified using both morphological and molecular characterization. For

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morphological identification, fungal isolation technique, previously described was used.15 The

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PDA plate was inoculated using a sterile needle. A sterile glass coverslip was placed in oblique

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position on the culture and plates were incubated at 25 °C for 4-6 d. From day 2 onwards,

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coverslips were removed daily and mounted on a slide stained with lactophenol aniline blue

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(Sigma-Aldrich, St. Louis, MO). The coverslips were examined using a light microscope (Leica,

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Wetzlar, Germany) at the magnification ×8 and ×40.

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Molecular characterization of MI-1 was initiated by isolation of genomic DNA using CTAB

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method from mycelia grown on PDA plates.16 PCR was conducted using internal transcribed

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spacers (ITS) primer pairs.17 Non-specific PCR products generated, were further subjected to

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band-stab PCR.18 The PCR comprised of a 25 µL reaction consisting of PCR buffer (1x), ITS1

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primer (0.2 µM), ITS4 primer (0.2 µM), dNTPs (0.06 mM), MgCl2 (2 mM), Taq polymerase (0.5

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U) (Thermo Scientific, Wyman, MA). The primer sequences for ITS1 and ITS4 were 5'

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TCCGTAGGTGAACCTGCGG 3' and 5' TCCTCCGCTTATTGATATGC 3', respectively.17

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This reaction mix was subjected to the following PCR program: initial denaturation 94°C for 5

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min, followed by 40 cycles of denaturation 94 °C for 45 s, annealing temperature 60 °C for 30 s,

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elongation 72 °C for 90 s and a final elongation of 72 °C for 6 min. PCR was conducted on

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several other microbes to verify that the amplicon generated from MI-1 was not present in other

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microbes or if present, it was not of a similar size. The microbes included were as follows: MI-1;

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Metarhizium anisopliae IC30 from ICIPE; Ceratocystis fimbriata f. sp. platani (CBS 127659);

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Clavibacter michiganensis ssp. michiganensis; Catharanthus roseus containing Udinese-Stolbur

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phytoplasma from CBS-KNAW Fungal Biodiversity Centre (Utrecht, the Netherlands) and an

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unknown fungus culture collected from potato fields were grown on PDA media containing

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streptomycin sulfate. PCR products were resolved on 1% agarose gel in TBE for 1 h at 100V.

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PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany)

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and Gel Extraction kit (Qiagen, Hilden, Germany). Purified PCR products were sequenced using

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GS-FLX 454 platform (454 Life Sciences/Roche, Bradford, CT).

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Extraction and Identification of Furanoterpenoids from Fungal-Infected Sweetpotatoes

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The storage root samples were prepared using a previously described method2 with few

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modifications. Healthy storage roots from sweetpotato cultivars namely Kemb, Naspot, Nyawo

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and Bungoma were washed, surface sterilized for 5 min using 0.5% sodium hypochlorite and

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rinsed 3 times in sterile distilled water in a laminar flow hood. In the first experiment, the storage

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root samples were cut into halves and placed in clean sterile plastic containers before inoculating

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them with actively growing fungal isolate (MI-1) from agar plugs while the controls were not

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inoculated. The sweetpotato samples (both inoculated and non-inoculated) were replicated three

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times for each cultivar. The second experiment consisted of inoculating Kemb and Naspot

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cultivars using MI-1 and the storage roots were tied with polythene and elastic bags to restrict

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growth of the fungus. The storage roots were incubated at 25 °C for 7-14 d for infection. This

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second experiment consisted of 2 cultivars, Kemb and Naspot with 7 samples. Each sample was

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chopped into 1-cm slices to analyze furanoterpenoid levels from infection point to the healthy-

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looking tissue. Differences between means were calculated using Student Newman Keuls test.

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The fungus covering sweetpotato were scraped off. The storage roots were then weighed and

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blended in 100 mL methanol and 3g NaCl for 3 min. For isolation and purification of

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dehydroipomeamarone and ipomeamarone, 268g of infected sweet potato was extracted with 550

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mL of methanol. The extracts were filtered using Whatman filter paper No. 4, poured in conical

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flasks and then concentrated in a rotary evaporator to remove methanol and water. The volume

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of concentrated crude extract was estimated as 10 mL and the same volume of 10 mL

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dichloromethane (Sigma, St. Louis, MO) was added to effectively double the volume of the

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extract for each flask. The organic phases were combined, concentrated to dryness in a rotary

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evaporator to remove any remaining solvents from the crude extract, weighed and then purified

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by chromatography.

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Fifteen grams of the extracted materials were chromatographed on 32-63 µm silica gel (Riedel-

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de Haen, Seelze, Germany) using a hexane-ethyl acetate gradient. The furanoterpenoids fraction

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eluted with 90% hexane: ethyl acetate which was further cleaned by column chromatography

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using a hexane-acetone gradient to yield ipomeamarone mix (1g). Further purification of the 7



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eluent was carried out on a Shimadzu VP series using a 205 mm x 10 mm i.d., 5 µM ACE Q C-

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18 column (ACE, Aberdeen, Scotland). The mobile phase used an isocratic program (A:B),

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60:40 with a flow rate of 1 mL/min and run time was 20 min. Detection was by UV absorption at

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270 nm to obtain 18 mg ipomeamarone and 15 mg dehydroipomeamarone with percentage yield

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being 0.12% and 0.10%, respectively (Figure 2). Structures of the isolated furanoterpenoids were

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determined by means of chromatographic and spectroscopic techniques (LC-QToF-MS, GC-MS

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and 1- and 2-D NMR) and by comparison with spectroscopic literature data.1, 19

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For GC-MS analyses, the furanoterpenoids fractions was analyzed by split/splitless injection

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using a model 7890 gas chromatograph coupled to a 5975C inert XL EI/CI mass spectrometer

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(Agilent technologies, Palo Alto, CA) (GC-MS) equipped with a 30 m × 0.25 mm i.d. × 0.25 µm

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film thickness HP-5 column (El 70eV) (Agilent technologies, Palo Alto, CA). Helium was used

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as the carrier gas at a flow rate of 1.2 mL/min. The oven temperature was held at 35 °C for 3

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min, then programmed to increase at 10 °C/min to 280 °C and maintained at this temperature for

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10 min. The target peaks were identified through comparison of their mass spectra with

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Adams2.L, Chemecol.L and NIST05a.L library data (Figure 3).

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For LC-QToF-MS analyses, the crude extract was concentrated in vacuo to dryness and then re-

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dissolved in 3 mL LC-MS grade CHROMASOLV methanol (Sigma-Aldrich, St. Louis, MO),

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centrifuged at 14,000 rpm for 5 min after which 0.5 µL was automatically injected into LC-

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QToF-MS. The chromatographic separation was achieved on a Waters ACQUITY UPLC (ultra-

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performance liquid chromatography) I-class system (Waters Corporation, Maple Street, MA)

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fitted with a 2.1 mm × 100 mm, 1.7-µm particle size Waters ACQUITY UPLC BEH C18

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column (Waters Corporation, Dublin, Ireland) heated to 40 °C and an auto sampler tray cooled to

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15 °C. Mobile phases of water (A) and acetonitrile (B), each with 0.01% formic acid were

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employed. The following gradient was used 0-1.5 min, 10% B; 1.5-2 min, 10−50% B; 2-6 min,

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50-100% B; 6-9 min, 100% B; 9-10 min, 90-10%; 10-12 min, 10% B. The flow rate was held

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constant at 0.4 mL/min. The UPLC system was interfaced by electrospray ionization (ESI) to a

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Waters Xevo QToF-MS operated in full scan MSE in positive mode. Data were acquired in

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resolution mode over the m/z range 100-1200 with a scan time of 1 s using a capillary voltage of

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0.5 kV, sampling cone voltage of 40 V, source temperature 100 °C and desolvation temperature

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of 350 °C. The nitrogen desolvation flow rate was 500 L/h. For the high-energy scan function, a

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collision energy ramp of 25-45 eV was applied in the T-wave collision cell using ultrahigh purity

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argon (≥99.999%) as the collision gas. A continuous lock spray reference compound (leucine

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enkephalin; [M+H] +=556.2766) was sampled at 10 s intervals for centroid data mass correction.

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The mass spectrometer was calibrated across the 50-1,200 Da mass range using a 0.5 mM

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sodium formate solution prepared in 90:10 2-propanol/water (v/v). MassLynx version 4.1 SCN

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712 (Waters Corporation, Maple Street, MA) was used for data acquisition and processing. The

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elemental composition was generated for every analyte. Potential assignments were calculated

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using mono-isotopic masses with specifications of a tolerance of 10 ppm deviation and both odd-

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and even-electron states possible. The number and types of expected atoms was set as follows:

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carbon ≤ 100; hydrogen ≤ 100; oxygen ≤ 50; nitrogen ≤ 6; sulfur ≤ 6. The LC-Qtof-Ms data

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acquisition and analysis was based on the following defined parameters: Mass accuracy (ppm) =

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1,000,000 × (calculated mass − accurate mass)/calculated mass; Fit conf % is the confidence

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with which accurate mass (measured data) matches the theoretical isotope models of the

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elemental composition in the list; Elemental composition is a suggested formula for the specified

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mass. This is a summation of the quantities of elements, isotopes, and/or super-atoms that can

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compose the measured data, calculated using the following atomic masses of the most abundant

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isotope of the elements: C = 12.0000000, H = 1.0078250, N = 14.0030740, O = 15.9949146, and

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S = 31.9720718. The empirical formula generated was used to predict structures which were

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proposed based on the online database, fragmentation pattern and literature.

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In order to verify the identity of these peaks, nuclear magnetic resonance (NMR) was done. The

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isolated and purified ipomeamarone and dehydroipomeamarone samples (5 mg each) were each

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dissolved in CDCl3 (Cambridge Isotope Laboratories, Tewksbury, MA) and placed in 2.5-mm x

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100-mm MATCH NMR tubes (Norell, Landisville, NJ). 1D and 2D 1H and

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spectroscopy, including correlation spectroscopy (COSY), heteronuclear single-quantum

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coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC) spectroscopy, was used

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for verification. For the dehydroipomeamarone, 2D nuclear Overhauser effect spectroscopy

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(NOESY) data was collected and analyzed for additional structural verification. All NMR

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spectra were acquired at 22 °C using a 5-mm TXI CryoProbe (Bruker Corporation, Billerica,

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MA) and a Bruker Avance II 600 console (600 MHz for 1H and 151 MHz for 13C) except for 2D

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NOESY data which was collected at 25°C. Residual CHCl3 was used to reference chemical shifts

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to δ 7.26 ppm for 1H, and δ C1 of ipomeamarone is referenced to 72.6 ppm for 13C in the HSQC

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spectrum by a previous report for consistency with the literature.20 We also checked the residual

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CHCl3 in CDCl3 for

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according to a previous study.21 NMR spectra were processed using Bruker Topspin 2.0 and

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MestReNova (Mestrelab Research) software packages.

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Quantitation of Furanoterpenoids in Sweetpotato Samples

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LC-QToF-MS in full scan MSE in positive mode was used to detect furanoterpenoids in extracts

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based on accurate mass measurement, retention time, fragmentation pattern and reference spectra

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database published online of isolated dehydroipomeamarone and ipomeamarone (standards).22

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C NMR

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C in the HSQC spectra to confirm that it was properly referenced

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Dehydroipomeamarone and ipomeamarone were quantitated using generated standard calibration

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curves prepared from the isolated compounds. Serially diluted solutions of isolated standards

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(0.01-200 µg/µL) were analyzed by LC-QToF-MS to generate linear calibration curves (peak

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area

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dehydroipomeamarone [y=324980x+177617 (R2= 0.9995)] which served as the basis for

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external quantitation.

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Results and Discussion

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Identification of MI-1 as Rhizopus stolonifer

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Identification of MI-1 fungus isolated from the field-collected sweetpotato storage roots was

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conducted because different microbes elicit varying furanoterpenoid responses.2 Based on

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morphological features (sporangia, sporangiophores, spores, collumella, rhizoids and stolons);

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sporangiophores arising from intersections with rhizoids and stolons; the dome-shaped columella

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and not falling off when the sporangium dried out, MI-1 was identified belonging to the genus

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Rhizopus. Morphological features of MI-1 had similarities to previously reported R. stolonifer.23,

233

24

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species as previously reported as follows: R. oryzae has an ellipsoidal collumella; R. sexualis, a

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conical-cylindrical shape and R. microspores, a sub-globose to conical shape.25 From these

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observations, it was appropriate to suggest that MI-1 fungus was R. stolonifer.

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The morphological features of Rhizopus further confirmed by molecular evidence enabled us

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identify the species. The ITS primer pair generated an amplicon of 950 bp for MI-1 fungus; none

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of the other species in this study had a similar band size as MI-1. Metarhizium anisopliae IC30

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had a band of 600 bp. Sequencing generated a number of reads but searches were conducted

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using sequences longer than 200 bp, based on non-redundant database of NCBI, producing 100

vs.

concentration)

for

ipomeamarone

[y=583064x+642221

(R2=0.9991)]

and

The major finding was the collumella being dome-shaped, which differed from other Rhizopus

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hits. These hits were mainly ITS1, ITS2, 5.8S, 18S and 28S fragments with partial or complete

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length sequences; the top 19 hits based on e-value were Rhizopus stolonifer. They had sequence

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similarity of 91-99% with e-values between 0 to 9×10-162. The best alignment to MI-1 was isolate

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AM933546.1 having a sequence identity of 98% and an e-level of 0. The R. stolonifer isolate had

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a partial sequence of 18S rRNA gene, ITS1, 5.8S rRNA gene, ITS2 and partial sequence of 28S

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rRNA gene. Molecular characterization using ITS primers confirmed that MI-1 fungus was R.

248

stolonifer: such primers have previously been used in R. stolonifer identification.16

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Chromatographic and Spectroscopic Techniques Confirmed Presence of Furanoterpenoids

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The furanoterpenoids ipomeamarone and dehydroipomeamarone were successfully identified

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using HPLC, GC-MS, LC-QToF-MS, 1D and 2D NMR techniques by comparing their

252

resonances to published data.20,

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ipomeamarone and dehydroipomeamarone eluting at 16.4 and 11.6 min, respectively. GC-MS

254

analysis also revealed 6 peaks, and tentatively identified 2 of these peaks as ipomeamarone, 1, at

255

a retention time (Rt) of 20.8 min and dehydroipomeamarone, 2, at Rt of 21.4 min (Figure 3). LC-

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QToF-MS analysis also identified the 2 peaks as ipomeamarone (with elemental composition,

257

C15H23O3; m/z 251.1647, 0.0 ppm error to theoretical value and a fit conf % of 99.96) at a

258

retention time (Rt) of 2.87 min and dehydroipomeamarone, (with elemental composition,

259

C15H21O3; m/z 249.1491, 2.0 ppm error to theoretical value and a fit conf % of 98.78) at a

260

retention time (Rt) of 3.04 min. These results were consistent with GC-MS analysis.

261

The HPLC purified peaks analyzed by NMR confirmed the presence of ipomeamarone and

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dehydroipomeamarone. 1H chemical shifts of ipomeamarone was in agreement with literature

263

while dehydroipomeamarone was a mixture based on LC-QToF-MS and NMR data.20, 27 For the

21, 26, 27

The HPLC analysis (Figure 2) showed 6 peaks with

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dehydroipomeamarone, we observed the 1H chemical shifts at positions C-7 as 6.15 ppm (1H),

265

C-9 as 2.13 ppm (3H), C-10 as 1.85 ppm (3H) which were the only 1H chemical shifts as

266

provided by previous workers.27 These workers reported the 1H chemical shifts for positions C-7

267

as 6.11ppm (1H), C-9 as 2.09 ppm (3H), C-10 as 1.81 ppm (3H), and all of the other 1H chemical

268

shifts were similar to ipomeamarone.27 Unfortunately, these three 1H chemical shifts differed

269

0.04 ppm between our spectrum and previously reported data.27 Since the dehydroipomeamarone

270

sample was a mixture, having just 1H 1D NMR was not satisfactory to confirm the presence of

271

dehydroipomeamarone. Unfortunately, previous workers did not provide any information about

272

NMR solvent or 13C chemical shift data for the synthetic dehydroipomeamarone.27 Therefore, we

273

acquired a 2D NMR (COSY, HSQC, HMBC, NOESY) spectrum for dehydroipomeamarone and

274

ipomeamarone samples to compare the

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derivative of ipomeamarone, which we confirmed using 1H NMR. Ipomeamarone 2D NMR data

276

was used for comparison. Both 1H and

277

similar to ipomeamarone for carbon positions from 1' to 4' and from 1 to 6, but differed at

278

positions 7, 8, 9 and 10: consistent with the structure. NOESY data for dehydroipomeamarone

279

provided us additional evidence to confirm dehydroipomeamarone hence identification.

280

Variations in Ipomeamarone Concentrations between and within Cultivars

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The main furanoterpenoids produced were ipomeamarone, 1, and dehydroipomeamarone, 2,

282

found in all samples and non-inoculated controls. The concentration of furanoterpenoid ranged

283

between 50.6-2,330 mg/kg for inoculated samples while 12.4-144.5 mg/kg for the controls.

284

These high levels exceeded those reported previously for R. stolonifer of 200-1,100 mg/kg but

285

comparable to high ipomeamarone elicitors such as C. fimbriata and Fusarium solani with levels

286

from 1,100-9,300 mg/kg.2 Similar results have been reported on non-infected sweetpotatoes with

13

C chemical shifts, since dehydroipomeamarone is a

13

C chemical shifts of dehydroipomeamarone were

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low ipomeamarone concentrations between 40-325 mg/kg for cultivars in the United Kingdom.8

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Generally, mechanical injury or damaged but non-infected sweetpotato is reported to elicit low

289

furanoterpenoid levels while infected samples have high levels due to increase in enzyme

290

activity, correlating to furanoterpenoid production.28, 29 The low ipomeamarone levels in storage

291

roots of negative control samples from this study confirm that injury or bruising of sweetpotato

292

roots occurs but the ipomeamarone levels are low.

293

Concentration levels for ipomeamarone ranged between 50.6-2126.7 mg/kg while 39.3-2,230.4

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mg/kg for dehydroipomeamarone. The higher dehydroipomeamarone levels could possibly be

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because it is a precursor of ipomeamarone, which meant that it was yet to be enzymatically

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converted to ipomeamarone or dehydroipomeamarone might have inhibited production of

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ipomeamarone to some extent as reported previously.1 In preliminary experiments (not shown),

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ipomeamarone levels were high in inoculated samples, probably due to long incubation time of

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28 d compared to the current study where inoculation ranged between 7-14 d. More research

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needs to be conducted to verify this.

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Variations in mean ipomeamarone levels for the cultivars were as follows: Kemb had the highest

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mean 1,476.2 mg/kg followed by Nyawo with 1,089.9 mg/kg, Naspot had 833.7 mg/kg while

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Bungoma had 676.5 mg/kg (Table 1). High concentrations of dehydroipomeamarone were also

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observed in almost all samples in this study: Kemb had the highest with 1,462.6 mg/kg, Nyawo

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had 1,459.7 mg/kg, Naspot had 1,153.2 mg/kg while Bungoma had 910 mg/kg (Table 1). There

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were significant (p

Toxic Ipomeamarone Accumulation in Healthy ... - ACS Publications

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