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. 2026 Sep 16;12(9):691. doi: 10.3390/jof12090691

Development of a PCR Assay Targeting the iboH Gene to Assess the Distribution of Ibotenic Acid/Muscimol Biosynthesis Potential in the Genus Amanita

Zheng-Mi He 1,*,†, Jia-Xi Li 1,†
Editor: Sotiris Amillis1
PMCID: PMC13608680  PMID: 42783986

Abstract

Ibotenic acid and muscimol, synthesized by the ibotenic acid biosynthetic gene cluster (ibo BGC) in Amanita species, are two of the primary neurotoxins responsible for mushroom poisoning incidents worldwide. However, the molecular detection methods for these toxins remain underdeveloped. In this study, we designed a pair of universal PCR primers targeting iboH, a hydroxylase-encoding gene within the ibo BGC, and developed a PCR assay with a detection limit of 10 pg of genomic DNA per reaction. Using the assay on 53 Amanita species, 21 out of the 23 species tested from section Amanita were positive, whereas all 30 species from sects. Amidella (1), Caesareae (3), Phalloideae (6), Roanokenses (8), Strobiliformes (1), Vaginatae (4), and Validae (7) tested negative. We then integrated these PCR signals into our global phylogenetic analysis of Amanita and found that iboH is restricted to a large clade within sect. Amanita. Amanita sinensis, commonly considered an edible species in China, was detected carrying iboH, indicating its potential to produce ibotenic acid/muscimol and pose associated health risks. In conclusion, this study establishes an effective universal PCR assay for detecting ibotenic acid/muscimol-producing Amanita species and offer a reference for toxigenic mushroom recognition and food safety.

Keywords: ibotenic acid, muscimol, molecular detection, mushroom poisoning

1. Introduction

Amanita (Amanitaceae, Agaricales) is a cosmopolitan genus comprising 11 sections and over 650 species [1,2]. It is well-known for its edible species but even more notorious for its poisonous ones [3]. Among them, section Amanita, with 112 accepted species reported [2,4,5,6,7], is one of the major groups responsible for neurotoxicity in mushroom poisoning worldwide [8]. This section is characterized by an agaricoid basidioma that has a pileus bearing a striate margin and persistent volval remnants on its surface, a stipe with basal bulb, and inamyloid basidiospores [9,10]. People may easily mistake some poisonous species of this section for other edible Amanita mushrooms like A. caesarea, A. hemibapha, and A. rubescens. Indeed, species of this section have caused numerous poisoning incidents, even resulting in fatalities [11,12]. In Europe and North America, A. muscaria and A. pantherina are the species most frequently reported [13,14], whereas in China, the main species involved include A. melleiceps, A. rufoferruginea, A. subglobosa, and A. sychnopyramis [15]. From 2019 to 2024, a total of 147 poisoning incidents involving 492 patients related to these species were documented in China [16,17,18,19,20,21], which highlights the urgent need for rapid and reliable identification and risk assessment methods.

The primary neurotoxins in sect. Amanita are ibotenic acid (IBO) and muscimol (MUS), two isoxazoles that are structurally analogous to glutamic acid and gamma-aminobutyric acid (GABA) [22]. Both toxins can stimulate N-methyl-D-aspartate (NMDA) and GABA receptors, thereby inducing central nervous system symptoms, such as dizziness, somnolence, delirium, dysphoria, and hallucinations [23]. These compounds were initially isolated from A. muscaria [24,25] and have since been detected in 15 other species of this section [6,15,26,27,28,29,30,31]: A. altipes, A. concentrica, A. cothurnata, A. flavopantherina, A. gemmata, A. griseopantherina, A. ibotengutake, A. pantherina, A. parvisychnopyramis, A. pseudopantherina, A. pseudosychnopyramis, A. regalis, A. rubrovolvata, A. subglobosa, and A. sychnopyramis.

The biosynthesis pathways of IBO and MUS were recently elucidated by Obermaier and Müller [32]. Their findings revealed that the synthesis of these compounds is derived from the amino acids glutamine or glutamate and involves seven core enzymes encoded by a biosynthetic gene cluster (BGC): IboA (adenylating), IboC (cytochrome P450), IboD (decarboxylase), IboF (flavin-dependent monooxygenase), IboG1 and G2 (two pyridoxal phosphate-dependent enzymes), and IboH (hydroxylase). Su et al. [33] identified this ibo BGC in 21 Chinese species that form a major clade within sect. Amanita, suggesting a monophyletic distribution of the BGC and a widespread genetic capacity for IBO/MUS production within this section. However, whether the ibo BGC is present in other sections of Amanita has not been sufficiently investigated through extensive sampling.

Currently, liquid chromatography–mass spectrometry (LC-MS) is the conventional method for detecting IBO and MUS [29,34]. This method can be applied in the detection of mushroom or human urine samples for the investigation of food poisoning cases [35,36]. However, it requires expensive instrumentation, chemical standards, and professional personnel, thus impeding its application in large-scale or routine screenings. In comparison, PCR-based molecular assays offer distinct advantages: (1) PCR instruments and reagents are far more affordable, and the workflow is simpler, which is particularly advantageous in resource-limited settings; (2) PCR allows multiplexed analysis and typically 96 samples to be processed in a single run, whereas LC-MS processes samples individually, which drastically reduces throughput and becomes time-consuming for large-scale analyses. However, to date, there has been no simple and rapid molecular assay available to infer the presence of these toxins based on their biosynthetic genes.

In our previous work [33], we retrieved the ibo BGC genes from 21 Amanita species, including 21 iboA, 19 iboC, 20 iboD, 19 iboF, 19 iboG1, 18 iboG2, and 18 iboH, thereby laying the foundation for the design of specific primers. In this study, we selected the 18 iboH sequences obtained from our previous work [33] as target templates for primer design, because that iboH is a 620-bp, intron-free, and highly conserved gene indispensable for glutamate hydroxylation in the IBO/MUS biosynthesis. Based on these iboH sequences, we designed specific primers and developed a universal PCR-based method targeting iboH. Applying this method to 53 Amanita species from eight sections, we investigated the occurrence and distribution of iboH across the genus Amanita. This PCR-based method provides a simple, rapid, and cost-effective alternative to LC-MS, with applications in clinical diagnostics and prevention, food safety monitoring, and epidemiological surveillance of mushroom poisoning.

2. Materials and Methods

2.1. Fungal Specimens and Identification

A total of 53 Amanita specimens collected from China were used in this study. Detailed information for each specimen is provided in Table 1. Fresh basidiomata (Figure 1) were photographed in situ using a Sony ILCE-7 camera (Sony Inc., Tokyo, Japan). Specimens were dried at 45 °C using a fruit dehydrator (KD728, Kakool, Guangzhou, China) and accessioned at the Mycological Herbarium of Hunan Normal University (MHHNU, Changsha, China). Specimens were identified based on morphological characters using the monographs of Yang et al. [9,10] and Cui et al. [1]. Identifications were further verified by DNA barcode sequencing and phylogenetic analysis as described below.

Table 1.

Species and specimens included in phylogenetic analysis and PCR assay, with examined specimens and newly generated sequences in this study in bold.

Section Species Voucher ITS LSU iboH IBO/MUS
Amanita A. albocreata RET 547-7 KU248128 ✓ [37]
A. alpinicola MONT CLC2376 NR158316 [38]
A. altipes HKAS 3660 AY436445 [39]
A. altipes MHHNU 33050 C_AA120855 [33] C_AA120982 [33] +, PZ793121 ✓ [15]
A. ‘amerlpanthera’ RET 387-10 MT445432 HQ539707
A. aprica RET 128-5 NR154683 [40] NG057029 [40] * [40]
A. ‘austrowellsii’ RET 302-1 MN963578 MN963578
A. bingensis KM 104 MT446264 [41] MT446281 [41]
A. breckonii NY 00066695 KJ535439 KJ535440
A. cf. albopulverulenta KM 48 MT446282 [41]
A. chrysoblema RET 320-1 EU071911 [42] EU071984 [42] ✓ [43]
A. collariata MHHNU 31095 OM955206 [5] OM955204 [5] +, PZ793122 * [5,20]
A. concentrica CBM FB-24901 NR119387 [44] ✓ [15]
A. crenulata RET 675-9 MK204468 MK204474 + [32] ✓ [45]
A. cruzii BARONI 8998 KC855222 [46] KC855222 [46]
A. diemii MES 1335 KY053387 [47] E [48]
A. digitosa BBH 32154 KT213722 [49] ✓ [50]
A. elata HKAS 83449 MH508334 [1] MH486486 [1]
A. farinosa MHHNU 32693 ON131732 [15] ON139027 [15] - × [15]
A. farinosa MHHNU 32768 ON131733 [15] ON139028 [15]
A. fibrillopes PERTH08793573 MN918103 [51] MN918099 [51]
A. flavoalba CAL 1405 KY861748 [52]
A. flavomelleiceps MHHNU 33119 PQ326880 PQ330908 +, PZ793123
A. flavopantherina MHHNU 32267 ON131735 ON139030 +, PZ793124 ✓ [15]
A. flavopantherina HKAS 82613 MH508355 [1] MH486519 [1]
A. frostiana RET 543-6/RET 7-25-92 E KP313580 AF024453 [53] ✓ [54]
A. gemmata JM96/62 AF097371 [55] ✓ [26]
A. gioiosa ML61952AG MH603599 [56]
A. gloeocystidiosa BBH 31903 KT213719 [49]
A. griseopantherina HKAS 83560 MH508385 [1] MH486573 [1]
A. griseopantherina MHHNU 33084 C_AA120883 [33] C_AA121009 [33] +, PZ793125 ✓ [15]
A. ‘hallingiana’ RET 292-7 KY924833
A. ibotengutake CBM FB-30969/30974 NR119388 [28] AB088767 [28]
A. kalasinensis CMUB-39966 OM040562 [4] OM040553 [4]
A. melleialba HKAS 83446 MH508430 [1] KR824767 [1]
A. melleialba MHHNU 10606 C_AA120884 [33] C_AA121010 [33] +, PZ793126 * [18]
A. melleiceps MHHNU 31865 ON131765 [15] ON139065 [15] +, PZ793127 * [19,20,21]
A. melleiceps MHHNU 32763 ON131767 [15] ON139067 [15]
A. minima KM 38 MT446261 [41] MT446280 [41]
A. mira HKAS 91953 MH508437 [1] MH486646 [1] * [57]
A. multisquamosa RET 374-1/BW RP14 MK840916 [58] HQ539710 ✓ [37]
A. muscaria MHHNU 33653 C_AA120885 [33] C_AA121011 [33] +, PZ793128 ✓ [15,24]
A. muscaria MHHNU 31764 ON131738 ON139033
A. nehuta JAC10720 MT863749 MT862256
A. orientigemmata HKAS 80978 MH508469 [1] MH486708 [1]
A. orientigemmata MHHNU 32700 ON131764 [15] ON139063 [15] +, PZ793129 * [17,18,21]
A. pakistanica RET 317-6 KX365198 KX365199
A. pantherina MB-102863 MH508488 [1] MH486743 [1] + [32] ✓ [29]
A. pantherinoides iNAT 25463910 OM212868 ✓ [59]
A. parcivolvata RET 614-4 MN963585 MN963584
A. parvipantherina HKAS 83663 MH508499 MH486752
A. parvipantherina MHHNU 32942 C_AA120859 [33] C_AA120986 [33] +, PZ793130 * [20,21]
A. parvisychnopyramis MHHNU 32953 PQ326876 [6] PQ330907 [6] +, PZ793131 ✓ [6]
A. persicina RET 151-4 EU071892 [42] EU071969 [42] ✓ [37]
A. ‘praecox’ BW-PH082906-9 HQ539725 ✓ [37]
A. pseudopantherina HKAS 80007 MH508514 [1] MH486777 [1]
A. pseudopantherina MHHNU 11333 C_AA120861 [33] C_AA120988 [33] +, PZ793132 ✓ [15]
A. pseudosychnopyramis HKAS 87999 MH508530 [1] KR824778 [1]
A. pseudosychnopyramis MHHNU 32762 MZ313998 [15] OM753110 [15] +, PZ793133 ✓ [15]
A. pudica RET 344-40 HQ539730
A. pubescens RET 802-10 MH930991 MH931002
A. pyriformis BBH 38643 KT213723 [49]
A. ravicrocina CMUB 39967 OM040568 [4] OM040559 [4]
A. regalis HKAS 56699 MH508537 [1] MH486797 [1] ✓ [31]
A. rubrovolvata MHHNU 32524 ON131751 [15] ON139046 [15]
A. rubrovolvata MHHNU 32949 C_AA120864 [33] C_AA120991 [33] +, PZ793134 ✓ [15]
A. rufoferruginea HKAS 84974 MH508580 [1] MH486843 [1]
A. rufoferruginea MHHNU 31100 ON131773 [15] ON139073 [15] +, PZ793135 ✓ [15]
A. robusta YA KM 41 NR173187 [41] MT446278 [41]
A. roseotincta RET 313-5 MH508551 [1]
A. siamensis HKAS 67855 MH508592 [1] MH486864 [1]
A. siamensis MHHNU 31578 ON131776 [15] +, PZ793136 * [20,21,60]
A. sinensis HKAS 80012 MH508597 [1] MH486871 [1]
A. sinensis MHHNU 8585 MK239263 [15] ON139047 [15] +, PZ793137
A. stranella TENN 60935 FJ596814 [61]
A. subfrostiana HKAS 32513 AF024477 [53]
A. subfrostiana MHHNU 32955 C_AA120873 [33] C_AA120999 [33] +, PZ793138
A. subglobosa HKAS 12009 AF024478 [53]
A. subglobosa MHHNU 32538 ON131753 [15] ON139051 [15] +, PZ793139 ✓ [15]
A. submelleialba CMUB S1 NR184934 [62] NG088232 [62]
A. subparcivolvata MHHNU 32368 OM955213 [5] OM955211 [5] - × [15]
A. subparcivolvata MHHNU 32907 OM955216 [5] OM955205 [5]
A. subparvipantherina HKAS 56986 KR824776 [63]
A. subparvipantherina MHHNU 32226 ON131756 [15] ON139054 [15] +, PZ793140 +
A. ‘subvelatipes’ RET 483-10 MK204467
A. sychnopyramis MHHNU 32153 ON131761 [15] ON139060 [15]
A. sychnopyramis MHHNU 33330 C_AA120879 [33] C_AA121005 [33] +, PZ793141 ✓ [15]
A. taiepa JAC12826 MT863755 MT862263
A. ‘tlaxcalipanthera’ MO 428571 MW633035
A. velatipes RET 489-2 MH508643 [1] MH486938 [1]
A. viscidolutea ANMF 767 MW000473
A. wellsii RET 259-1 KU248116 OK285331
A. xylinivolva ANDES F312 NVE56 FJ890024 [64] FJ890036 [64]
Amarrendia A. oleosa H7627 GQ925400 [65] GQ925377 [65]
A. umbrinella PSC 1813 AY194981 HQ539753
Amidella A. rufobrunnescens GDGM 42374 KT865208 [66] KT865210 [66]
A. rufobrunnescens MHHNU 33470 PZ832437 -
A. volvata S. Harsch 304 AF024485 [53]
Arenariae A. arenaria VPI679 GQ925382 [65]
Caesareae A. caesarea HKAS 96166 MH508283 [1] MH486418 [1] E [67]
A. kitamagotake HKAS 107309B MW258874 [68] MW258921 [68]
A. kitamagotake MHHNU 33459 PZ832438 PZ830472 - E [57]
A. princeps MHHNU 31274 PV564110 [69] PZ830473 -
A. princeps HKAS 122502 ON794332 [70]
A. yuaniana HKAS 68662 MH508654 [1] MH486957 [1]
A. yuaniana MHHNU 34553 PZ832439 PZ830474 - E [57]
Phalloideae A. exitialis HKAS 75775 JX998026 [71] JX998053
A. exitialis MHHNU 20088 PZ832440 -
A. fuliginea MHHNU 9047 MN061271 [72]
A. fuliginea MHHNU 34221 PZ832441 -
A. pallidorosea MHHNU 8112 KU311697 [73]
A. pallidorosea MHHNU10523 PZ832442 PZ830475 -
A. phalloides HKAS 75773 JX998031 [71] JX998060 [71]
A. rimosa MHHNU 7954 KU311695 [74]
A. rimosa MHHNU 11109 PZ832443 PZ830476 -
A. subjunquillea BJTC FM2244 PV265320 [75] PV259883 [75]
A. subjunquillea MHHNU 32832 PZ830477 -
A. subpallidorosea HKAS 85403 KP691683 [76] KP691692 [76]
A. subpallidorosea MHHNU 34182 PZ832444 -
Roanokenses A. atrobrunnea MFLU 15-1415 NG057090 [49]
A. atrobrunnea MHHNU 34151 PX699301 -
A. caojizong HKAS 79673 MH508291 [1] MH486429 [1]
A. caojizong MHHNU 32855 PZ830478 - E [57]
A. eijii HKAS 70229 MH486484 [1]
A. eijii MHHNU 10639 PZ830479 -
A. gymnopus MHHNU 33197 PX699306 -
A. gymnopus HKAS 71618 MH508393 [1] MH486582 [1]
A. kotohiraensis MHHNU 6998 FJ176722 [77] FJ011681 [77]
A. kotohiraensis MHHNU 33391 PX696944 PX699307 -
A. manginiana HKAS 101404 MH486637
A. manginiana MHHNU 34165 PX699310 [1] -
A. oberwinklerana BJTC FM3352 PV259872 [74]
A. oberwinklerana MHHNU 34779 PZ830480 -
A. rubiginosa HKAS 52216 MH508561 [1] MH486817 [1]
A. rubiginosa MHHNU 9701 PZ830481 -
Strobiliformes A. griseoverrucosa HKAS 38459 AY436486 [1] AY436495 [1]
A. griseoverrucosa MHHNU 20089 PZ830482 -
A. strobiliformis MB-001177 MH508614 [1] MH486895 [1]
Vaginatae A. brunneofuliginea HKAS 83536 MH508268 [1] MH486392 [1]
A. brunneofuliginea MHHNU 33664 PZ832445 -
A. changtuia HKAS 92100 NR159572 [1] NG064562 [1]
A. changtuia MHHNU 20090 PZ832446 -
A. hamadae HKAS 79076 MH508394 [1] MH486584 [1]
A. hamadae MHHNU 34767 PZ832447 -
A. umbrinolutea HKAS 89637 MH508638 [1] MH486934 [1]
A. umbrinolutea MHHNU 34330 PZ832448 PZ830483 -
Validae A. brunneolimbata HKAS 78459 MH508274 [1]
A. brunneolimbata MHHNU 33436 PZ832449 PZ830484 -
A. excelsa HKAS 31510 AY436453 [39] AY436491 [39]
A. flavoconia HKAS 87990 MH508345 [1] MH486508 [1]
A. flavoconia MHHNU 34414 PZ832450 PZ830485 -
A. pseudofritillaria MHHNU 34731 PZ832451 PZ830486 -
A. pseudofritillaria MHKMU T. Huang 398 ON768708 [78]
A. rubescens HKAS 100631 MH486810 [1]
A. rubescens MHHNU 34775 PZ830487 - E [79]
A. sepiacea HKAS 74862B MW258870 [69]
A. sepiacea MHHNU 34722 PZ832452 PZ830488 -
A. sinocitrina HKAS 83445 MH508601 [1] MH486876 [1]
A. sinocitrina MHHNU 34746 PZ832453 PZ830489 - × [80]
A. submaculata P19FSMF MW589098 [81]
A. submaculata MHHNU 32842 PV564112 [69] PZ830490 -
Lepidella A. flavofloccosa HKAS 90174/92006 MH508352 [1] MH486516 [1]
A. vittadinii HKAS 101430 MH508651 [1] MH486950 [1]
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“+”, species confirmed to contain iboH; “-”, species confirmed to lack iboH; blank, no data or not confirmed; “✓”, species reported to contain IBO/MUS; “×”, species reported to lack IBO/MUS; “E”, species reported to be edible; “*”, species with documented human poisoning cases presenting symptoms consistent with IBO/MUS exposure.

Figure 1.

Figure 1

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Basidiomata of representative Amanita species from different sections. Section Amanita: (a) A. altipes (MHHNU 33050); (b) A. farinosa (MHHNU 32693); (c) A. parvipantherina (MHHNU 32942); (d) A. pseudosychnopyramis (MHHNU 32762); (e) A. rufoferruginea (MHHNU 31100); (f) A. sinensis (MHHNU 8585); (g) A. subfrostiana (MHHNU 32955); (h) A. subglobosa (MHHNU 32538); (i) A. subparvipantherina (MHHNU 32226); sect. Amidella: (j) A. rufobrunnescens (MHHNU 33470); sect. Caesareae: (k) A. princeps (MHHNU 31274); (l) A. yuaniana (MHHNU 34553); sect. Phalloideae: (m) A. exitialis (MHHNU 20088); (n) A. pallidorosea (MHHNU 10523); sect. Roanokenses: (o) A. caojizong (MHHNU 32855); (p) A. eijii (MHHNU 10639); (q) A. gymnopus (MHHNU 33197); (r) A. manginiana (MHHNU 34165); (s) A. oberwinklerana (MHHNU 34779); sect. Vaginatae: (t) A. umbrinolutea (MHHNU 34330); sect. Validae: (u) A. brunneolimbata (MHHNU 33436); (v) A. flavoconia (MHHNU 34414); (w) A. rubescens (MHHNU 34775); (x) A. submaculata (MHHNU 32842). Scale bars = 1 cm.

2.2. DNA Extraction, Primer Design, PCR Amplification, and Sequencing

Total genomic DNA was extracted using the Fungal DNA Mini Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s instructions. Universal PCR primers targeting iboH were designed based on a sequence alignment of iboH genes from 18 Amanita species reported by Su et al. [33] (Figure 2). The designed primer pair was IboH-F (5′-CTYGTYCTTCTYCAYTACCCR-3′, 21 bp) and IboH-R (5′-SCGRTCTTSYTCYTGGTA-3′, 18 bp), yielding a predicted amplification product of 279 bp. The specificity of the primers was further verified using Primer-BLAST v2.5.0 [82]. The internal transcribed spacer (ITS) was amplified using the primer pair ITS5/ITS4 [83], and the nuclear ribosomal large subunit (LSU) was amplified using LR0R/LR5 [84]. These primers were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China).

The PCR mixture was prepared in a total volume of 25 μL, consisting of 1× Hieff® PCR Master Mix (Yeasen Biotechnology, Shanghai, China; providing 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, and 1.25 U of Taq polymerase), 0.4 μM forward primer, 0.4 μM reverse primer, and 1 μL of DNA template. PCR was performed in an ABI 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA). The thermal cycling program consisted of an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C (LSU), 54 °C (ITS) or 56 °C (iboH) for 30 s, and extension at 72 °C for 30 s (ITS and iboH) or 60 s (LSU), with a final extension at 72 °C for 10 min.

PCR products were examined by electrophoresis on a 2% agarose gel in 1× TAE buffer (Solarbio, Beijing, China) at 110 V for 15 to 20 min. A DL2000 DNA marker (BioRun, Wuhan, China) was used as the molecular weight standard. Gels were photographed under a JP-2880 gel documentation system (Jiapeng, Shanghai, China). The successful amplicons were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing.

2.3. Specificity and Sensitivity of PCR Assay Targeting iboH

To evaluate the specificity of the specific PCR primers IboH-F/IboH-R designed above, genomic DNA from 53 Amanita species representing eight sections of the genus (Table 1) was tested. For sect. Amanita, 18 species had been confirmed to carry iboH and two species (A. farinosa and A. subparcivolvata) had been confirmed to lack iboH by Su et al. [33]. Three sect. Amanita species (A. flavomelleiceps, A. sinensis, and A. pseudopantherina) had not been previously confirmed. The status of iboH in the remaining 30 tested species from seven other sections were unknown. For each PCR run, ddH2O was used as the blank control in place of DNA template, and all samples were tested in at least three independent replicates to ensure reproducibility.

To determine the limit of detection (LOD), the PCR tests were performed using serial 10-fold dilutions of the genomic DNA from A. altipes (MHHNU 33050) ranging from 1 μg to 1 pg.

2.4. Identification of iboH Genes

Chromatograms from bidirectional sequencing of iboH amplicons were visualized using BioEdit v7.2.5 [85] to check peak quality and to confirm that these nucleotide sequences (excluding primers) were 240 bp in length. The trimmed nucleotide sequences were translated into amino acid sequences and aligned with the IboH sequence of A. muscaria Koide BX008 (KIL56739) using DNAMAN v7.212 (Lynnon Biosoft, Vaudreuil-Dorion, QC, Canada) for identification.

2.5. Phylogenetic Analysis

To achieve a comprehensive and high-coverage phylogenetic analysis, sequences of 112 representative Amanita species encompassing all 11 sections of the genus were included (Table 1). Among these, sequences of 53 species were obtained from our Chinese specimens, while sequences of the remaining 59 species, originating from Africa, Europe, North America, Oceania, and other regions of Asia, were downloaded from GenBank (https://ncbi.nlm.nih.gov; accessed on 7 March 2026). ITS and LSU sequences (Table 1) were aligned using MAFFT v7.511 [86]. Ambiguously aligned regions were trimmed using Gblocks v0.91b [87]. The two loci were concatenated using SequenceMatrix 1.7.8 [88]. The generated dataset for Amanita comprised 1414 positions from 163 samples (Supplementary Material S1).

According to the AIC criterion in MrModeltest v2.4 [89], GTR + I + G was estimated as the best-fit model for the ITS and LSU. Bayesian inference (BI) analysis was performed in MrBayes v3.2.7 [90] using the selected model, with two simultaneous runs, four Markov Chain Monte Carlo (MCMC) chains, and sampling every 1000 generations over a total of 5,000,000 generations, until the average standard deviation of split frequencies fell below 0.01. The first 25% generations were discarded as burn-in, and convergence was visually assessed using the trace function in Tracer v1.7.2 [91]. Maximum likelihood (ML) analysis with 1000 bootstrap replicates was computed in RAxML v8.0.20 [92] using the GTR + G + I model.

Figure 2.

Figure 2

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Multiple sequence alignment of iboH genes from 18 Amanita species, showing the conserved region (nucleotide positions 101–500) used for primer design. Binding sites of the forward and reverse primers are indicated by arrows. Sequences were downloaded from GenBase [93].

3. Results

3.1. PCR Assay Targeting iboH and Sequence Identification

To detect the occurrence of the iboH gene in Amanita, DNA samples extracted from 53 Amanita species belonging to eight sections were tested. The electrophoretic results are shown in Table 1 and Figure 3a. Overall, 21 species tested positive, yielding bands slightly larger than 250 bp (expected size: 271 bp), while the remaining 32 species and the no temple control tested negative. At the section level, positive reactions occurred only in sect. Amanita [21/23], whereas negative reactions occurred in all other sections: Amidella [0/1], Caesareae [0/3], Phalloideae [0/6], Roanokenses [0/8], Strobiliformes [0/1], Vaginatae [0/4], and Validae [0/7].

Figure 3.

Figure 3

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PCR detection of iboH in Amanita species. (a) Agarose gel showing amplification results from representative species; (b) alignment of the obtained sequences with the reference IboH sequence from A. muscaria Koide BX008 (GenBank: KIL56739) over the region spanning positions 63–142. M, DL 2000 DNA marker; lanes 1–23, sect. Amanita (names in red); lane 24, sect. Amidella; lane 25–27, sect. Caesareae; lane 28–33, sect. Phalloideae; lane 29–41, sect. Roanokenses; lane 42, sect. Strobiliformes; lane 43–46, sect. Vaginatae; lane 47–53, sect. Roanokenses; lane 54: no template control (NTC).

Sequencing results (Table 1) further showed that all positive amplicons were 240 bp in length after removing the primer regions (21 bp for IboH-F and 18 bp for IboH-R), which was consistent with the expected size. The deduced amino acid sequences aligned well with the template IboH sequence from A. muscaria Koide BX008 (GenBank accession no. KIL56739) [32], showing 0–5 amino acid differences (Figure 3b). These results validate the identity of the positive amplicons and confirmed the specificity of the designed primers.

To determine the detection limit, the PCR was performed using a serial 10-fold dilution ranging from 1 μg to 1 pg of DNA template of A. altipes. The electrophoresis result (Figure 4) showed that the detection limit of the PCR assay was 10 pg of genomic DNA per reaction.

Figure 4.

Figure 4

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Sensitivity of PCR assay targeting iboH. M, DL 2000 DNA marker; NTC, no template control. A ten-fold dilution series of genomic DNA of A. altipes was tested: lane 1, 1 μg; lane 2, 100 ng; lane 3, 10 ng, lane 4, 1 ng; lane 5, 100 pg; lane 6, 10 pg; lane 7, 1 pg.

3.2. Phylogenetic Distribution of iboH Within Amanita

Our global phylogenetic analysis of Amanita (Figure 5) recovered 112 species. Among them, our Chinese specimens were confirmed to represent 53 known species. The analysis also recovered 11 sections: Amanita (97% BP, 1.00 PP), Amarrendiae (100% BP, 1.00 PP), Amidella (100% BP, 1.00 PP), Amidella, Caesareae (100% BP, 1.00 PP), Phalloideae (100% BP, 1.00 PP), Roanokenses (90% BP, 1.00 PP), Strobiliformes (81% BP, 1.00 PP), Vaginatae (100% BP, 1.00 PP), and Validae (94% BP, 1.00 PP). The section Amanita was further divided into three major clades and two species lineages: the IBO clade (42% BP, 1.00 PP), Clade B (88% BP, 1.00 PP), Clade C (99% BP, 1.00 PP), and the A. diemii and A. taiepa lineages.

Figure 5.

Figure 5

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ML analysis of Amanita based on concatenated ITS-LSU sequence data. Bootstrap values (BP) ≥ 50% from ML analysis and Bayesian posterior probabilities (PP) ≥ 0.90 from BI analysis are shown at nodes. Geographic origins of specimens are presented, with type specimens in parentheses and Chinese specimens from this study in bold. Clades A, B, and C of Su et al. [33] are indicated by arrows. Red and green lines indicate the presence and absence of iboH/IBO/MUS, respectively. Blue, red, and pink circles to the right of the tree indicate species with genomic, chemical, and poisoning evidence for IBO/MUS, respectively, while white circles indicate species without iboH/IBO/MUS. “E” indicates edible species, while black lines and circles indicate lack of data or unconfirmed. For the iboH gene, data sourced from Obermaier and Müller [32] are marked with asterisks (*), while all other iboH data were generated in the present study. IBO/MUS detection data and poisoning case records were compiled from previously published studies, with the corresponding citations provided in Table 1.

As shown in Figure 5, all 21 iboH-positive species, together with the IBO/MUS-containing species and those previously reported to cause human poisoning with symptoms consistent with IBO/MUS exposure, fell within the IBO clade of sect. Amanita. In contrast, all 32 iboH-negative species, along with species reported to lack IBO/MUS and edible species were all recovered outside this clade.

4. Discussion

4.1. ibo BGC Distribution Within Amanita and Delimitation of the IBO Clade

In our previous work [33], we identified the ibo BGC in 20 known Amanita species by shallow whole-genome sequencing, but failed to detect iboH in A. flavomelleiceps and A. pseudopantherina. In the present study, we revisited these species using PCR and successfully confirmed the presence of iboH in all 20 species. We additionally detected the gene in A. sinensis. Given that iboH is a core gene within the ibo BGC [32], the presence of this gene implies the existence of the complete BGC in these species. The BGC was also found in A. crenulata and A. pantherina [32]. Another 11 species, viz. A. albocreata [37], A. concentrica [15], A. digitosa [50], A. frostiana [54], A. gemmata [26], A. ibotengutake [28], A. multisquamosa [37], A. pantherinoides [59], A. persicina [37], A. praecox [37], A. regalis [31], and A. velatipes [37], have been reported to contain IBO/MUS, and two additional species, A. aprica [40] and A. mira [57], have been implicated in poisonings with symptoms consistent with IBO/MUS exposure. These observations suggest that the ibo BGC is present in all 36 species listed above. Interestingly, all these species fall into a large monophyletic clade within sect. Amanita, viz., the IBO clade (Figure 5).

By contrast, iboH was not detected in 32 species, which included representatives from Clades B (1) and C (1) of sect. Amanita, as well as from sects. Amidella (1), Caesareae (3), Phalloideae (6), Roanokenses (8), Strobiliformes (1), Vaginatae (4), and Validae (7). Two additional species, A. diemii [48] and A. caesarea [67], have been reported to be edible. Therefore, these 34 species are considered to lack the ibo BGC and as expected, all of them fall outside the IBO clade (Figure 5).

Collectively, these findings indicate that the ibo BGC is restricted to a large clade within sect. Amanita, suggesting it has an independent origin within Amanita. By extending our sampling from sect. Amanita in Su et al. [33] to the entire genus using a PCR approach, we corroborate the earlier report by Su et al. [33] using genomic sequencing and further demonstrate that this restriction is maintained across the genus.

The IBO clade is defined here as the monophyletic group encompassing all Amanita species that (i) harbor ibo genes, (ii) have been chemically confirmed to produce IBO/MUS, or (iii) are associated with poisoning cases exhibiting symptoms typical of IBO/MUS intoxication. In the ITS-LSU phylogenetic analysis of Su et al. [33], sect. Amanita was resolved into three clades, designated as Clades A–C, of which Clades B and C received strong support, whereas Clade A was only weakly supported. In our ITS-LSU analysis (Figure 5), Clades B and C of Su et al. [33] also received strong support, but Clade A of Su et al. [33] received no statistical support. However, after excluding A. diemii and A. taiepa from Clade A, the remaining species formed a strongly supported clade with 1.00 PP in BI analyses of Su et al. [33] and the present study. Therefore, Clade A as a whole is unstable, while the IBO clade is more likely corresponds to this well-supported clade. Salazar Vidal [48] reported that A. diemii has been consumed by the Mapuche people and rural communities in Chile for over a century, which supports our exclusion of this species from the IBO clade. We therefore recognize this clade as the IBO clade, as shown in Figure 5. A more precise delimitation of this clade will require using more genetic loci in phylogenetic analysis and more comprehensive ibo gene detection.

4.2. Considerations of Edibility and Toxicity of Amanita sinensis

Amanita sinensis is generally recorded as an edible species in the literature [94,95] and monographs [9,10], and is known to be consumed locally in Southern China. However, a recently published monograph on Chinese poisonous mushrooms [96] classifies this species as a gastrointestinal irritant, and reports a case in Yunnan Province in which ingestion led to palpitations and nausea in the patients. In this study, we successfully detected iboH in this species and confirmed its identity by sequencing (Figure 3b). This finding indicates that this species is highly likely to harbor the ibo BGC for the production of IBO/MUS. Accordingly, we consider that consumption of A. sinensis may present potential human health risks.

4.3. Specificity and Universality of the iboH-Targeted PCR Primers

The iboH-targeted PCR primers show high universality, successfully amplifying the target gene from 20 Amanita species previously confirmed to possess the ibo BGC [33], with no amplification from A. farinosa and A. subparcivolvata. These two species were confirmed to lack all genes of the ibo BGC, including iboH, by genome sequencing conducted by Su et al. [33], which is consistent with our PCR results. Despite interspecific iboH differences, all tested iboH-containing species were consistently detected (Figure 3a). The specificity was further validated by Sanger sequencing, which confirmed the identity of all products as iboH (Figure 3b). Notably, a very faint band of approximately 100 bp was observed for A. subjunquella (lane 32, Figure 3a), which represents a non-specific amplification. However, this product is clearly distinct from the expected 271 bp iboH amplicon and given its low intensity, it did not interfere with the visual interpretation or downstream sequencing validation.

4.4. Comparison Between PCR and LC-MS for Detection of IBO/MUS

LC-MS detects the toxins IBO and MUS themselves, providing direct identification and quantification of the actual chemical compounds present in samples. It is applicable to diverse matrices including mushrooms [97], human urine [98], serum [99], and gastric contents [100], making it the gold standard for clinical diagnosis and forensic investigation of poisoning cases. However, the technique requires expensive instrumentation and chemically standards, well-trained personnel, and complicated sample preparation and experiment operation. Additionally, IBO and MUS are chemically unstable and prone to degradation [15,101], which may lead to false-negative results.

Our PCR-based assay, which targets the iboH gene associated with IBO/MUS biosynthesis, exhibits significant advantages over LC-MS. PCR instruments and reagents are approximately 100-fold cheaper than those for LC-MS, and the experimental workflow is simpler, making it more suitable for primary hospitals and centers for disease control (CDC). Although a single PCR run usually takes about 1 h (vs. usually 0.5 h per sample for LC-MS), PCR allows simultaneous processing of multiple samples, whereas LC-MS operates in a single-sample sequence. Moreover, DNA is more stable than IBO/MUS in fungal specimens [15,33], enabling PCR successful detection even after toxin degradation.

In the present study, we employed PCR-based detection for iboH as a molecular screening tool to assess the toxigenic potential of 53 representative Amanita species across eight sections of the genus. When we cross-referenced our PCR results with previously published LC-MS data and poisoning case reports presenting with IBO/MUS-like symptoms (Table 1 and Figure 5), we found that the concordance between PCR-based findings and chemical/clinical evidence reached 100%. The overall concordance between the presence of iboH and the presence of IBO/MUS supports the utility of our PCR-based assay for assessing toxigenic potential.

Our PCR-based assay also has inherent limitations that must be acknowledged. Most critically, PCR detects the presence of the iboH gene rather than the toxin itself, a positive result indicates toxigenic potential but does not confirm that toxins are actually being produced or accumulated in detectable quantities. This method provides no quantitative information about toxin concentration, which is often crucial for assessing poisoning severity.

Despite these limitations, the PCR-based assay established in this study offers a low-cost, rapid, and operationally simple alternative that is well-suited for primary hospitals in preliminary poisoning screening, as well as for local CDC laboratories in poisoning investigation and food risk assessment. In future work, the development of isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and rolling circle amplification (RCA), could further reduce the reliance on thermal cycling equipment, improve operational convenience, and enable accessibility for on-site-detection.

Acknowledgments

We are grateful to Zuo-Hong Chen and Ping Zhang (Hunan Normal University) for collecting specimens and providing their images.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jof12090691/s1, Supplementary Material S1: ITS-LSU sequence alignment.

jof-12-00691-s001.zip (18.9KB, zip)

Author Contributions

Conceptualization, primer design, image editing and enhancement, and manuscript writing, Z.-M.H.; DNA extraction, PCR, electrophoresis, species identification, sequence data, and phylogenetic analysis, J.-X.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The DNA sequences obtained in this study have been submitted to GenBank.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This study was financially supported by the National Natural Science Foundation of China (Grant No. 32400010) and Hunan Province Major Basic Research Projects (Grant No. 2026JC0008).

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jof-12-00691-s001.zip (18.9KB, zip)

Data Availability Statement

The DNA sequences obtained in this study have been submitted to GenBank.

Articles from Journal of Fungi are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

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