Fusarium elaeidis Causes Stem and Root Rot on Alocasia longiloba in South China

Alocasia longiloba is a popular ornamental plant in China, however pests and diseases associated with A. longiloba reduce the ornamental value of this plant. From 2016 to 2021, stem and root rot has been observed on A. longiloba in Guangdong Province, China. Once the disease became severe, plants wilted and died. A fungus was isolated from the diseased stem and identified as Fusarium elaeidis using both morphological characteristics and molecular analysis of DNA-directed RNA polymerase II subunit (rpb2), translation elongation factor-1α (tef1) gene and β-tubulin (tub2) sequence data. The pathogenicity test showed the fungus was able to produce typical symptoms on A. longiloba similar to those observed in the field. The original pathogen was reisolated from inoculated plants fulfilling Koch’s postulates. This is the first report of Fusarium elaeidis causing stem rot on A. longiloba. These results will provide a baseline to identify and control diseases associated with A. longiloba.


Introduction
The ornamental plant industry has become a high demanding industry with the increased interest in modern architecture. The values of ornamental plants are based on the appearance of the plant. However, they are susceptible to several diseases including wilts, rots and leaf spots which reduce the economical values of the plants [1]. In addition, the ornamental plant industry is the main global source of planting material exchanges. Thus, it is possible to introduce a new pathogen to a new locality via these planting materials which can affect already established plants as well [2] Therefore, the identification and characterisation of phytopathogenic genera on ornamental plants are crucial.
Four Alocasia species are grown in China: A. cucullata, A. hainanica, A. longiloba and A. macrorrhiza. Alocasia longiloba is a perennial herb belonging to the Araceae (L). It is native to tropical Asia and in China this plant is mainly distributed in Hainan, Yunnan and Guangdong provinces. Among these A. longiloba is widely cultivated in Guangdong Province as an ornamental plant due to its distinctive 'elephant ear' foliage. There are several diseases and pests associated with Alocasia spp., including fungal diseases [3,4]. Alternaria alocasiae and Ceratocystis fimbriata have been reported to cause leaf spots [4] and leaf blight [5], respectively, on A. macrorrhiza in China [4]. In addition, anthracnose of A. macrorrhiza caused by Colletotrichum karstii [3], root rot on Alocasia sp. caused by Pythium sp. and Rhizoctonia solani in Florida, USA [6] are among reported diseases. Most of these studies were based on both morphological and phylogenetic aspects [3]. However, so far there are no records of fungal diseases on A. longiloba [7].
Fusarium is one of the well-known phytopathogenic genera that belong to the Nectriaceae [8,9]. In addition, they are also saprobes, endophytes, soil-borne or can be isolated from water [10]. Fusarium species are cosmopolitan, and the traditional classification of these fungi was based on morphology which has led to controversial arguments for many years. Species belonging to F. solani species complex were transferred to the Neocosmospora [11,12], however, this was not accepted by O'Donnell et al. 2020 [13] who suggested that the F. solani species complex should remain as a species complex within the Fusarium [13]. Species delineation in Fusarium is based on morphological characters and molecular phylogeny [14]. In the Fusarium taxonomy and molecular phylogeny, the rDNA fragments; the nuclear ribosomal internal transcribed spacer (ITS) and large subunit (LSU) are uninformative in species-level identification [15,16]. However, the tef1 is highly informative at the species level [16]. In addition, DNA-directed RNA polymerase II subunit RPB1 (rpb1) and rpb2 are also informative for species identification in Fusarium [14,17].
During 2016-2021, a new stem and rot rot disease was observed on A. longiloba in Guangzhou City (Guangdong Province, China). This study aims to identify the causal organism of this disease. Based on both morphological and molecular approaches, the pathogen was identified as Fusarium elaeidis. The pathogenicity of this species was confirmed on potted A. longiloba plants.

Field Symptoms
In the field, a one pot usually contains three to four plants. In these pots one or two plants showed wilt symptoms. The disease begins with a progressive leaf yellowing, followed by wilting, until the whole plant collapsed at the end (Figure 1a). Roots of diseased plants became black and rotted (Figure 1b). When diseased plants were cut open longitudinally the inside of the stems appeared brown (Figure 1c). Disease incidence was as high as 10% in some nurseries in Guangzhou City. In the phylogenetic tree, isolates from A. longiloba were grouped with F. elaeidis strains with 97% ML bootstrap support and 1.00 BYPP.
Notes: Isolates obtained from this study formed a well-supported clade with the representative strains of Fusarium elaeidis (CBS 217.49; Ex-type strain and CBS 255.52). Most of the morphological characteristics in this study were similar to the original description of Fusarium elaeidis by Lombard et al. [18]. However, the isolates obtained in this study differed by 3-septate and 4-septate of macroconidia. Moreover, in this study isolates produce larger macroconidia than those of the original description, and no polyphialides were observed in this study. The base pairs of both rpb2 and tub2 in this study are same as that of F. elaeidis (strain no?). Only three base pairs of tef1 (totally 616 bp) were different with that of F. elaeidis (?) in Lombord et al. study [18]. Therefore, we identified our strains as F. elaeidis based on both morphology and molecular phylogeny.

Pathogenicity Test
The pathogenicity of isolates ZHKU 21-0005 and ZHKU 21-0006 was determined by inoculating healthy A. longiloba with mycelium suspension. Leaves of inoculated plants turned yellow after ten days (Figure 4a). Root and stem showed black and started to rot after fifteen days of inoculation (Figure 4b turned yellow after ten days (Figure 4a). Root and stem showed black and started to rot after fifteen days of inoculation (Figure 4b,c). The root density of inoculated plants was lower compared to healthy plants. Symptoms on the inoculated plants were similar to those on diseased plants in the field. None of the control plants developed symptoms (Figure 4d-f). From the diseased plants, the pathogen was reisolated. The colonies and morphological characteristics of isolates from inoculated plants were the same as those from the field.

Discussion
Fusarium is an important genus including large numbers of phytopathogenic species. Therefore, correct species identification is important for disease diagnosing and control. Twenty monophyletic species complexes have been reported in Fusarium [18]. The morphological species of F. oxysporum was recognized as a species complex because of its high level of phylogenetic diversity [18][19][20][21]. Laurence et al. [21] divided 17 independent evolutionary lineages into two phylogenetic species using genealogical concordance

Discussion
Fusarium is an important genus including large numbers of phytopathogenic species. Therefore, correct species identification is important for disease diagnosing and control. Twenty monophyletic species complexes have been reported in Fusarium [18]. The morphological species of F. oxysporum was recognized as a species complex because of its high level of phylogenetic diversity [18][19][20][21]. Laurence et al. [21] divided 17 independent evolutionary lineages into two phylogenetic species using genealogical concordance phylogenetic species recognition (GCPSR) criteria. Moreover, Lombard et al. [18] identified fifteen cryptic taxa using five gene regions in the Fusarium. Multi-genes analysis, such as ITS, IGS rDNA (the nuclear ribosomal intergenic spacer region), mating-type genes (mat1-1-1α-box), tef1 and tub2, are used to delineate species in Fusarium and beyond species level within the F. oxysporum species complex [2,20,[22][23][24]. In this study, we combined morphological characteristics with DNA sequence analysis of rpb2, tef1 and tub2 genes to ensure species identification accurately.
Fusarium oxysporum species complex is a well-established and diverse complex in this genus. It can infect around 150 plant species [10], including vegetables, fruits, and ornamental plants, and cause severe diseases. Among these F. oxysporum f. sp. cubense which is the causal agent of banana wilt is one of the well-know and destructive disease on banana worldwide [25]. Fusarium fabacearum infect Malay apple plants (Syzygium malaccensis) and caused tree death in Brazil in 2017 [25]. On ornamentals, the disease caused by F. oxysporum cause wilt, crown rot and root rot. Moreover, these hosts included several valuable ornamental plants, such as Cymbidium spp., Chrysanthemum spp., and Gladiolus spp. [1]. Fusarium wilt is a destructive disease in cyclamen production. This disease was Pathogens 2021, 10, 1395 7 of 11 the first outbreak in Germany and then quickly spread to all European production regions resulting in a huge effect on the cyclamen industry [2,26]. Fusarium wilt on Cymbidium spp. is a well-known disease that leads to stem rot and plant death [27,28]. Fusarium oxysporum can infect various cultivars of Cymbidium and hybrid Cymbidium. The disease incidence was up to 30% in some plantations in Guangdong, China [28]. Over 30 ornamental plant genera have been recorded as hosts of F. oxysporum [1]. In the present study, we isolated and identified F. elaeidis from A. longiloba causing stem and root rot.
Fusarium oxysporum f. sp. elaeidis was upgraded to the species level as Fusarium elaeidis by Lombard et al. [18]. In that study, they resolved 15 cryptic taxa which were named as forma specialis in Fusarium oxysporum [18]. Because of this, almost all the previous reports on this species are under Fusarium oxysporum f. sp. elaeidis. This species has been reported to cause Fusarium wilt in oil palm worldwide [29]. This pathogen is a soilborne fungus and difficult to control [30]. However, this is the first report of stem and root rot on A. longiloba caused by F. elaeidis. Therefore, further studies are necessary to understand the host range of this pathogen. Fusarium is a well-established genus with a larger number of forms and races. Given the same weight as other respective fields in mycology, identification of pathogenic species is the most critical step in plant pathology [31,32]. Moreover, in plant pathology, this identification is necessary to go beyond the species level. However, for most of the phytopathogenic fungal genera except like Fusarium, defining beyond species levels are not practised [32]. Even though lower-level ranking is well defined in Fusarium, in recent studies, some of these lower levels are upgraded into species level. Therefore, it is necessary to conclude how to delineate each species and define lower levels in Fusarium.

Fungal Isolation and Purification
Infected stems from diseased plants were first washed with running tap water to remove debris. Then the samples were cut into 0.5 × 0.5 cm sections including both healthy and diseased tissues. Those cuttings were surface sterilized by immersion in 75% ethanol for 10 s, 2.5% NaOCl for 40 s, and rinsed in sterile water three times. After that samples were dried in sterilized tissue paper. Dried cuttings were placed onto potato dextrose agar (PDA) and incubated at 25 • C. Pure cultures were obtained after three times hyphal tip isolations. In total four isolates were obtained. The cultures were deposited in the Culture Collection of Zhongkai University of Agriculture and Engineering (ZHKUCC 21-0003, ZHKUCC 21-0004, ZHKUCC 21-0005, ZHKUCC 21-0006, ZHKUCC 21-0007).

DNA Extraction, PCR Amplification and Sequencing
The genomic DNA of four isolates was extracted using a DNA rapid Extraction Kit (Aidlab Biotechnologies Co., Ltd, Beijing, China). A portion of DNA-directed RNA polymerase II subunit (rpb2), the translation elongation factor-1α gene (tef1) and the βtubulin genes (tub2) were amplified and sequenced. The tef1 region was amplified using primer pair EF-1H and EF-2T [33]. The tub2 region was amplified using primer pair T1 and CYLTUB1R [22,34]. The rpb2 region was amplified using primer pair 5f2 and 7cr [35,36]. PCR reactions were conducted in 25 µL volumes containing 12.5 µL of 2× Easy Taq PCR SuperMix (TransGen Biotech, Beijing, China), 1 µL DNA, each of 5µM premier (1 µL), ddH2O (9.5 µL). PCR amplification was performed with an initial denaturation step of Pathogens 2021, 10, 1395 8 of 11 94 • C for 5 min, followed by 35 cycles of 94 • C for 40 s, annealing at 55 • C for 45 s, and extension at 72 • C for 45 s, and a final extension at 72 • C for ten min. The PCR products were sequenced by Guangzhou Tianyi Huiyuan Science and Technology Co. Ltd (Guangzhou, China). For sequencing in both directions with forward and reverse primers were used. The DNA sequence of rpb2, tef1 and tub2 were deposited in the GenBank (Table 1).

Phylogenetic Analyses
Consensus sequences were derived from forward and reverse primer sequences for rpb2, tef1 and tub2, and compared against the GenBank database (https://blast.ncbi.nlm. nih.gov/Blast.cgi, accessed on 18 June 2021). Relevant sequence data of Fusarium species were obtained from the National Center for Biotechnology Information (NCBI) following Lomboard et al. [18]. Sequences were aligned using MAFFT version 7. Sequences were aligned manually using BioEdit where necessary. Concatenated dataset of rpb2, tef1, and tub2 were used for the phylogenetic analyses.
Phylogenetic analyses were conducted based on Maximum-likelihood in RAxML [37] and Bayesian analyses in MrBayes (v. 3.0b4) [38] The maximum likelihood analyses were conducted using RAxML-HPC2 on XSEDE (8.2.8) [39] in the CIPRES Science Gateway platform. The GTR+I+G evolution model was used with 1000 non-parametric bootstrapping iterations. Bayesian analysis was performed with six simultaneous Markov chains were run for 106 generations, sampling the trees at every 200th generation. From the 10,000 trees obtained, the first 2000 representing the burn-in phase were discarded. The remaining 8000 trees were used to calculate posterior probabilities (PPs) in a majority rule consensus tree. The final sequence alignment generated in this study was submitted to TreeBASE (https://treebase.org/treebase-web/home.html, accessed on 5 July 2021) under submission ID 28480.

Morphological Characterisation
Pure cultures incubated on PDA for 7 days at 25 • C under a 12 h light-dark cycle were used for colony characteristics and colonial growth rates. Cultures grown on carnation leaves agar (CLA) for 7-10 days at 25 • C under a 12 h light-dark cycle were used for microscopic characters observation. Morphological characters were photographed using an ECLIPSE 80i microscope (Nikon, Tokyo, Japan) and measurements were taken using NIS-Elements BR 3.2. Measurements of spore length and width of 50 spores were taken. The mean values were calculated with Microsoft Excel.

Pathogenicity Tests
Two representative isolates ZHKU21-0005 and ZHKU21-0006 were used to inoculate potted plants from the nursery. Representative isolates were incubated in PDA for three days to obtain mycelia. One gram of mycelium added into 100 mL sterilised water and blend to make into 3% mycelium suspension. Healthy plants were inoculated by pouring 50 mL of mycelium suspension into the substrate around the roots. Sterilised water (50 mL) was used as the control. For each treatment three replicates were used. All treated plants were maintained at 25 • C in a growth chamber. To fulfil Koch's postulates, the fungus was re-isolated from inoculated plants showing typical disease symptoms and compared to the original isolate.