First Report of Fruit Rot of Snake Fruit (Salacca zalacca) Caused by Thielaviopsis euricoi Based on Morphological and Phylogenetic Analyses

Abstract

Snake fruit (Salacca zalacca (Gaertner) Voss) is renowned for its taste and nutritional value; however, the snake fruit rot diseases caused by fungal pathogens can lead to significant economic losses. In this study, a fungal isolate was obtained from the rotten snake fruit with brown rot symptoms at the fruit apex upon removal of the peel. The causal agent was identified as Thielaviopsis species based on cultural and morphometric characteristics. Multi-locus phylogenetic analyses of the internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2) genes revealed that the isolates Te 5742 clustered within Thielaviopsis euricoi Bat. & A. F. Vital. Furthermore, pathogenicity tests fulfilled Koch’s postulates, confirming T. euricoi as the pathogen responsible for the observed rot. This is the first report of T. euricoi causing fruit rot on snake fruit worldwide and will provide information on monitoring and management of snake fruit diseases in the garden and market.

1. Introduction

Snake fruit (Salacca zalacca (Gaertner) Voss) is a prominent tropical fruit known for its distinctive sour and sweet flavor profile and high antioxidant activity, which exceeds that of other common tropical fruits such as mangosteen (Garcinia mangostana L.) and avocado (Persea americana Mill.) [1,2,3]. With the increasing global demand for fruit consumption, it holds significant potential for the international market [2,4]. However, the rising incidence of diseases, particularly fruit rots caused by pathogens such as Peniophora salaccae N. Suwannar., S. Boukaew, & J. Kumla, Thielaviopsis paradoxa (De Seynes) Höhn., and T. ethacetica Went, poses a serious threat to the sustainable development of the snake fruit industry. As reported, T. paradoxa was found as the predominant pathogen causing snake fruit rot in Indonesian traditional markets, and P. salaccae was identified as a novel causal agent of this disease [5,6]. The rotten symptoms are often observed at the fruit apex, while it remains unclear how the rot diseases initially happened.

The genus Thielaviopsis Went was established in 1893 based on T. ethacetica and accommodates the anamorphs of described Ceratocystis Ellis & Halst. species [7,8]. However, the sexual states of several species have not been observed, including T. euricoi (Bat. & A.F. Vital) A.E. Paulin, T.C. Harr. & McNew, T. musarum (R.S. Mitchell) Riedl, T. australis (J. Walker & Kile) Z.W. De Beer, T.A. Duong & M.J. Wingf., and T. neocaledoniae (Kiffer & Delon) Z.W. De Beer, T.A. Duong & M.J. Wingf. [7,9]. Discriminating between Thielaviopsis species based solely on morphology is challenging due to the absence of sexual structures in some species and the overlapping morphological characteristics among different species [9]. Therefore, multigene phylogenetic analyses based on ITS, tub2, and tef1-α gene sequences are indispensable for accurate species identification [8,9].

Thielaviopsis species are well-known soilborne pathogens affecting monocotyledonous plants. Symptoms caused by Thielaviopsis species are typically shown by necrosis tissue, the charcoal-like black appearance, and the bending of the terminal bud [10,11,12]. For instance, T. paradoxa and T. punctulata have been reported as the causal agents of black scorch in palm species (Arecaceae) [10,11]. T. paradoxa and T. ethacetica have been confirmed as pathogens causing fruit rot in snake fruit, calling people’s attention to the control of Thielaviopsis species in the snake fruit garden [5]. Notably, T. euricoi (syn. Hughesiella euricoi) was first described by Batista & A.F. Vital in 1956 from air samples [13]; to date, it has not been reported as a plant pathogen.

In the present study, we observed fruit rot on snake fruit caused by an isolate morphologically resembling T. euricoi. Given the limited information regarding the spectrum of pathogens affecting snake fruit, the aims of this study were to identify the causal agent using morphological and phylogenetic analyses methods and to demonstrate its pathogenicity on snake fruit. This study lays the groundwork for future disease management and informs the quarantine strategies of various countries aimed at preventing the introduction of this pathogen via snake fruit.

2. Materials and Methods

2.1. Sample Collection and Isolation

In April 2025, snake fruit (Salacca zalacca) carried by travelers entering via the port of Hong Kong, China, was intercepted by Guangzhou Customs in accordance with regulations prohibiting the import of plants by passengers. The specific origin of the fruit was undetermined. The samples were maintained at 4 °C and transported to the laboratory of the Guangzhou Customs Technology Center for further analysis. Once we received the snake fruit samples, we conducted the symptom examination and made records. The isolations were collected from symptomatic tissue and purified using the tissue isolation method [11]. The resulting pure cultures were deposited in Guangzhou Customs Technology Center (Guangzhou, China).

2.2. Morphological Identification and Characterization

Colony morphology and pigmentation were assessed on potato dextrose agar (PDA), malt extract agar (MEA), and oatmeal agar (OA) plates incubated at 25 ± 2 °C (12 h light/12 h dark cycle) for 7 days [14]. Morphological characteristics were examined and captured using a Leica M205 C stereomicroscope and a Leica K7 digital camera equipped with Leica Application Suite X (LAS X) software v.3.7.6 (Leica, Wetzlar, Germany). The dimensions of 50 conidia were measured for each isolate (n = 50). Additionally, synnemata observed on the surface of rotting snake fruit were examined and photographed.

2.3. Sequence Data

Genomic DNA of 7-day-old colonies was extracted using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. Polymerase chain reactions (PCR) were performed to amplify the internal transcribed spacer (ITS) region, the translation elongation factor 1-alpha (tef1-α), and the partial beta-tubulin (tub2) genes using primer pairs ITS5/ITS4, EF1F/EF2R, and Bt1a/Bt1b, respectively [15,16,17]. The PCR products were sequenced by Tianyi Huiyuan Biological Technology Co., Ltd. (Guangzhou, China). All sequences obtained in the study were deposited in GenBank (

Table 1. Details of fungal isolates used for phylogenetic analysis in this study.

Species Name	Culture Accession	Host	Country	GenBank Accession Numbers
				ITS	tef1	tub2
Ceratocystis cf. paradoxa	CBS 116770	Palm species	Ecuador	JX518334	JX518302	JX518366
	CBS 101054	Rosa sp.	Netherlands	JX518333	JX518301	JX518365
C. virescens	CMW 11164	Fagus americanum	USA	DQ520639	EF070413	EF070441
Thielaviopsis cerberus	CMW 35021	Theobroma cacao	Cameroon	JX518355	JX518323	JX518387
	CMW 36668	Theobroma cacao	Cameroon	JX518348	JX518380	JX518316
T. euricoi	CBS 893.70	NA	Brazil	JX518335	JX518303	JX518367
	CBS 107.22	Cocos nucifera	NA	JX518367	JX518304	JX518368
	Te 5742	Salacca zalacca	NA	PX736385	PX776694	PX776695
T. paradoxa	CMW 36642	Elaeis guineensis	Cameroon	JX518346	JX518314	JX518378
	CMW36686	Elaeis guineensis	Cameroon	JX518352	JX518320	JX518384
	CMW 36689	Elaeis guineensis	Cameroon	JX518342	JX518310	JX518374
T. radicicola	CMW 1032	Phoenix dactylifera	USA	KF612023	KF612024	KF612025
	CMW 26389	Lawsonia inermis	Mauritania	KF953932	KF953931	KF917202

Note: Newly generated sequences are shown in bold. NA, not applicable.

).

2.4. Phylogenetic Analyses

The sequences obtained for ITS, tub2, and tef1-α were aligned with reference sequences retrieved from GenBank (Table 1) using MEGA software v.7.0 with default settings. Maximum likelihood (ML) phylogenetic analysis was performed using MEGA v.6.0 (or 7.0/11.0) based on the GTR substitution model. Branch support was evaluated using 1000 nonparametric bootstrap replicates [11].

2.5. Pathogenicity Trials

Pathogenicity tests were conducted on healthy, surface-sterilized snake fruit incubated at 25 ± 2 °C with 90% relative humidity in the dark. Both conidial suspension and PDA plug inoculation methods were employed [11,12,18]. For inoculum preparation, isolate Te 5742 was cultured on PDA for 7 days. A conidial suspension was filtered by using sterile water through sterile gauze and adjusted to the concentration of 1 × 10⁵ conidia/mL [11].

For the suspension assay, 10 µL of the conidial suspension was dropped onto the surface of intact (unpeeled) fruit, with sterile water serving as the control [19]. Mycelial plugs (5 mm diameter) excised from the margin of 7-day-old colonies on PDA were placed onto the surface of both intact (unpeeled) and peeled snake fruit. Sterile PDA plugs were used as controls. Each treatment included three replicates consisting of 10 fruits each. Disease incidence (number of rotted fruits) and symptoms were recorded at 24, 48, and 72 h post-inoculation (hpi). To fulfill Koch’s postulates, the pathogen was re-isolated from symptomatic snake fruit tissues [11] and confirmed by morphological characteristics and tef1-α sequence analysis.

3. Results

3.1. Symptom Description and Morphological Characteristics

Intact snake fruit appeared asymptomatic (Figure 1A); however, brown rot was observed at the fruit apex upon removal of the peel. Fungal structures were visible near the residual stigma at the top of the fruit (Figure 1B,C). A total of four fungal isolates were obtained for their morphological similarity and purified from the rotten snake fruit tissue. Among these, isolate Te 5742 was selected as the representative strain for further characterization.

Isolate Te 5742 grew rapidly, covering the 90 mm petri dish within 48 h on PDA in 25 ± 2 °C. Colonies were initially hyaline to white, transitioning to fuscous black or greyish sepia with age, and sporulated abundantly on PDA, MEA, and OA (Figure 2A–C). A strong fruity aroma was noted on PDA cultures. Conidiophores were hyaline, flask, phialidic, and mononematous, exhibiting enteroblastic conidium ontogeny (Figure 2D). Primary conidia were hyaline, aseptate, and cylindrical (Figure 2D), measuring 8.59–11.80 × 4.04–5.49 μm (n = 50). Secondary conidia were produced holoblastically, aseptate, initially hyaline but becoming greyish sepia, and typically oblong with thick walls at maturity. Two distinct size ranges were observed for secondary conidia (Figure 2E): 9.79–12.67 × 5.69–8.81 μm and 15.29–23.28 × 6.57–12.05 μm (n = 50). Aleurioconidia (chlamydospores) were produced holoblastically, singly, or in short chains; these were dark mouse-gray, thick-walled, and subglobose to globose (Figure 2F), measuring 10.22–15.58 × 7.13–9.97 μm (n = 50). Synnemata observed on the host surface possessed a globose apex (Figure 2G). Ascomata were not observed.

3.2. Molecular Characterization and Phylogenetic Analyses

The ITS, tub2, and tef1-α sequences of isolate Te 5742 were deposited in GenBank (Table 1). The concatenated dataset consisted of 13 strains, with Ceratocystis virescens (CMW 11164) serving as an outgroup. Phylogenetic analysis revealed that isolate Te 5742 formed a well-supported clade with Thielaviopsis euricoi-type strains (CBS 893.70 and CBS 107.22) with 100% bootstrap support (Figure 3).

3.3. Pathogenicity Tests

Pathogenicity assays demonstrated that rot symptoms initiated on peeled snake fruit at 24 h post-inoculation (hpi) in both conidial suspension and PDA plug treatments. In contrast, intact (unpeeled) fruit remained asymptomatic at this stage (Figure 4A–C). By 48 hpi, peeled fruit treated with the conidial suspension was covered with profuse white mycelium, while that treated with PDA plugs exhibited dark conidial production (Figure 4D,E). Interestingly, symptoms on intact (unpeeled) fruit were delayed; mycelial growth was not observed until 72 hpi in the PDA plug treatment (Figure 4F). The control fruits remained symptomless (Figure 4G–I). The isolate Te 5742 caused rot symptoms in all inoculated snake fruits, and the disease incidences recorded at 24, 48, and 72 hpi were all 100%. The pathogen was re-isolated from the inoculated snake fruit and identified as T. euricoi based on consistent morphological characteristics and identical tef1-α sequences, thus fulfilling Koch’s postulates.

4. Discussion

Morphological identification within the genus Thielaviopsis is challenging due to overlapping sporulation characteristics. For instance, T. ovoidea (Nag Raj & W.B. Kendr.) A.E. Paulin, T.C. Harr. & McNew, T. populi (Veldeman ex Kiffer & Delon) A.E. Paulin, T.C. Harr. & McNew, and T. thielavioides are morphologically similar, distinguished only by minor differences in aleurioconidia [7]. Specifically, species within the Ceratocystis paradoxa complex (C. musarum, C. paradoxa, C. radicicola, and T. euricoi) exhibit limited diagnostic morphological differences. In the present study, a key diagnostic feature of isolate Te 5742 was the production of aleurioconidia either singly or in short chains, differentiating it from C. radicicola, where they are borne exclusively singly [9]. Consistent with previous descriptions [7,9], ascomata were not observed in our isolate. Notably, we observed secondary conidia in two distinct size ranges, a feature that has not been previously described for T. euricoi [7,9], suggesting potential intraspecific variation. Additionally, while many Ceratocystis species are known to emit intense fruity scents to attract insect vectors [20,21], this characteristic was not mentioned in the original description of T. euricoi by Mbenoun et al. [9].

Given these morphological ambiguities, multigene phylogenies are critical for the accurate identification of Thielaviopsis species. Previous studies have demonstrated that ITS, β-tubulin, and tef1-α gene sequences are effective in delimiting species complexes across the genus [9,11,12]. Our phylogenetic analyses placed isolate Te 5742 within the T. euricoi clade with high support, corroborating our morphological identification. These results further confirm the utility of combined DNA sequence data in revising phylogenetic relationships and defining species boundaries within Ceratocystis sensu lato [12].

The genus Thielaviopsis includes numerous economically important pathogens of monocotyledonous plants, such as pineapple (Ananas comosus), banana (Musa acuminata), and palms (Arecaceae) [8]. Historically, T. euricoi strains have been collected primarily from Arecaceae hosts, such as coconut palm (Cocos nucifera); however, its pathogenicity had not been previously confirmed. In this study, we isolated T. euricoi (strain Te 5742) as the new pathogen on rotting snake fruit. This finding aligns with the growing recognition of Thielaviopsis species as emerging threats to snake fruit production [5]. The initial symptom showed that the brown rot on the flesh was similar to previously reported snake fruit rot diseases caused by T. paradoxa and P. salaccae [5,6]. In the present study, the pathogenicity test results showed that peeled fruit was more easily infected by the pathogen. It can be explained by the protective role of the fruit peel, which acts as a physical barrier to impede fungal pathogens, and is also consistent with the previous research results [5]. Although not consistently observed, profuse white mycelium emerged on some snake fruit inoculated with PDA plugs after extended incubation (>72 hpi). We speculate that the abundant production of white hyphae is associated with spores actively utilizing host-derived nutrients for development. Until now, there is still limited information about how the Thielaviopsis species disperses and carries out infection on snake fruit. Targeted monitoring for the T. euricoi-caused rotten disease should be established in snake fruit orchards.

In conclusion, this is the first report worldwide of T. euricoi causing fruit rot on snake fruit. Although the precise geographical origin of the intercepted fruit could not be determined, the severe symptoms observed in our pathogenicity tests highlight a significant risk. The finding is a valuable addition to the existing knowledge about the fungal pathogens causing snake fruit rot, and the government should pay attention to the quarantine of imported snake fruit. The development of T. euricoi rapid detection technology in the future is necessary.

5. Conclusions

In this study, we identified Thielaviopsis euricoi as a new pathogen of a previously undescribed fruit rot on snake fruit. This conclusion is supported by morphological characterization, multi-locus phylogenetic analysis, and pathogenicity assays fulfilling Koch’s postulates. Our work constitutes the first global record of this fungal species as a pathogen of snake fruit. These findings provide a scientific foundation for future taxonomic studies, the development of disease management strategies for Thielaviopsis species, and the quarantine priorities in snake fruit trade.