Host Range and Loop-Mediated Isothermal Amplification Detection of Globisporangium sylvaticum from Guizhou, China

Globisporangium, especially G. sylvaticum, causes devastating root rot, blight, and other diseases in various species of cash crops. To investigate the distribution and host range of G. sylvaticum in Guizhou, a suitable habitat for this pathogen, we collected 156 root-diseased samples, isolated the pathogens, and found that G. sylvaticum is widespread and has eleven host plants, including four novel hosts. Furthermore, to effectively identify G. sylvaticum, we developed a simple and dependable method based on loop-mediated isothermal amplification (LAMP), which used a primer set designed from the internal transcribed spacer sequences with high specificity and sensitivity of 1 pg/μL. Additionally, to perform field identification, we used the “Plant-LAMP” method with crude DNA extraction to detect the pathogen in 45 root samples from nine species of plants. Our results showed that this method could effectively detect G. sylvaticum in diseased roots. Therefore, our findings not only enrich existing research on the diversity of pathogenic Globisporangium in Guizhou but also present an efficient LAMP field detection method that could significantly contribute to plant disease management and prevention.


Introduction
The genus Globisporangium is a recently described taxa that was segregated from Pythium [1,2]. Globisporangium is an important soil-borne and wide-host range plant pathogen distributed worldwide, including in the crop-growing regions of East Asia, Western Europe, North America, and Australia [3]. During hot and rainy seasons, Globisporangium oospores germinate readily and produce many sporangia and zoospores, which spread quickly through water or strong winds, resulting in various diseases [4]. Guizhou is a suitable environment for Globisporangium owing to its warm and humid climate, diverse and abundant vegetation, and multiple river systems [5]. Simple water conservation infrastructure and insufficient irrigation and drainage capacities are characteristic of the region's production base. Root rot and wilt diseases in vegetables and other vital crops occur frequently and spread quickly during seasons of constant rainfall and hot temperatures [6]. Several pathogenic Globisporangium species have been identified to be associated with root rot or damping-off of vegetables such as cabbage, ginger, green onions, and lettuce, with G. sylvaticum being especially prevalent.
Globisporangium sylvaticum is a heterothallic species originally isolated from soil in the United States and later discovered in China, Canada, Turkey, Germany, the Netherlands, and other countries [7][8][9]. Its pathogenicity and toxicity have since been examined in more detail. In Iowa and the Midwestern United States, the pathogen has already caused a large outbreak of soybean seed rot [10]. Globally, a large number of host plants have been

Morphological Studies
Globisporangium sylvaticum is heterothallic; therefore, two putative isolates with opposite mating types were incubated in the V8A medium for confrontation culture. The colony and sexual and asexual structures were observed under a microscope after 1-2 weeks of culture at 25 • C in the dark. A minimum of 20 measurements were randomly obtained for each structure using a light microscope. The isolates were morphologically identified based on the diagnostic keys in the "Monograph of the genus Pythium" edited by van der Plaats-Niterink (1981) [29].

DNA Extraction, PCR Analysis, and Multi-Locus Phylogeny
PrepMan Ultra Reagent was used to extract genomic DNA of all isolates from mycelia, as described by Baten et al. (2014) [30]. The DNA concentration was measured using a Nanodrop spectrophotometer (Thermo™ Fisher Scientific, Wilmington, DE, USA), and the DNA was diluted to 100 pg/µL for further use. Sequences of the ITS and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene regions were used for identification at the species level using the primer sets listed in Table S1 [31,32]. The PCR mixtures (total volume of 25 µL) were prepared using the TaKaRa Ex µTaq kit, and amplification was performed using a PCR device (846-x-070-723, Analytik Jena, Gottingen, Germany), as described in Table S1. Amplification products were separated using a 2% agarose gel (containing nucleic acid dye) and photographed under UV light. PCR products were sequenced by Sangon Biotech (Shanghai, China). The raw sequences were obtained and submitted to GenBank ( Table 1). The phylogenetic trees were generated using the Maximum Likelihood (ML) and Maximum Parsimony (MP) methods on the CIPRES web portal (https://www.phylo.org/portal2/login!input.action, accessed on 6 July 2023), utilizing the combined ITS and cox1 dataset. Elongisporangium dimorphum and E. prolatum were selected as outgroup. For the ML analysis, the RAxML-HPC BlackBox tool was employed with its default settings. On the other hand, the MP analysis was conducted using the PAUP 4.a168 tool on XSEDE.

Koch's Postulates
Pure G. sylvaticum isolates were cultured in conical flasks containing 100 mL of 10% V8 liquid medium and 50 autoclaved wheat seeds. The flasks were incubated at 150 rpm in the dark at 25 • C for 7 days. Budding wheat seedlings were transplanted into 0.4-L pots with sterilized substrate soil saturated with deionized water. The infection treatment involved placing eight infected wheat seeds near the roots of the seedlings, whereas the control treatment involved placing eight autoclaved seeds. Each group was comprised of five seedlings. All seedlings were planted in the artificial climate chamber (QHS-Z4Z), with a relative humidity of approximately 75% at 25 • C. After 28 days, the occurrence of the disease was observed and recorded, and the diseased roots were placed in the NARM medium.

LAMP and PCR Primer Design
The ITS region, which effectively distinguished G. sylvaticum from other species, except Pythium terrestris (G. terrestre), was selected as the target sequence for designing LAMP and PCR primers. Multiple alignments of different ITS sequences from G. sylvaticum and other Globisporangium species were analysed using the BioEdit Sequence Alignment Editor software ( Figure S1). Regions specific to G. sylvaticum were identified and used in primer design. In particular, the unique sequences were located at the 5 ends of LAMP primers-FIP (or F1c) and BIP (or B1c) and 3 ends of PCR primers. In addition, two-loop primers were added to accelerate the LAMP reaction. All LAMP and PCR primers were designed and analysed using PrimerExplorer V5 software (https://prime rexplorer.jp, accessed on 28 August 2022) or Primer3web (https://bioinfo.ut.ee/primer3/, accessed on 28 August 2022).  primers, 0.1 µM F-loop primer, and 1.6 µM FIP and BIP primers), 4.8 U of the Bst 2.0 DNA polymerase and template DNA (100 pg for specificity tests), and 0.2 µL of LAMP fluorescent dye. Specificity and sensitivity tests were performed to identify optimal primers. Furthermore, the reaction temperatures of 60, 62.5, 65, and 67.5 • C were tested as primary parameters to optimise the LAMP reaction. Reactions were conducted for 60 min in a 7500 Fast thermocycler (Applied Biosystems), and the real-time fluorescence intensities were recorded at intervals of 30 s, under a constant temperature of 65 • C. To visually evaluate the reaction results, the colour change of SYBR Green I dye was observed. Two drops of mineral oil were added to seal the reaction mixture before initiating the reaction. After the reaction, 2 µL of SYBR Green I (1000×) was added to the tube wall, sealed, and vortexed. Sterile distilled water was employed as a negative control, and genomic DNA from G. sylvaticum served as a positive control.

Specificity and Sensitivity of LAMP or PCR Primers in Detecting G. sylvaticum
All primer sets were first checked for specificity using 100 pg of genomic DNA template from two isolates of G. sylvaticum (GZso05 and GZco02) and seven genetically related isolates of the Globisporangium. One LAMP or PCR primer set with preliminarily determined specificity was further examined in a wide range of species. In total, 42 isolates comprising 25 Globisporangium, 9 Pythium, 2 Phytopythium, 3 Phytophthora isolates, and 3 isolates of other typical fungal pathogens were studied (Table 2). In addition, a series of 10-fold dilutions from 1 ng/µL to 1 fg/µL of G. sylvaticum (GZst02) genomic DNA were used for the sensitivity assay. The LAMP or PCR assay was performed using appropriate primers and reaction conditions as previously described. Table 2. Isolates used in this study for specificity testing of the LAMP and PCR primers.

Species
Clade Isolates a Origin Detection

Detection of G. sylvaticum in Plant Roots
The "plant LAMP (P-LAMP)" procedure, described by Feng et al. (2015), has been utilized to detect pathogens in various plant roots [33]. Rotting or browned roots were cut into 5 × 1 cm pieces, collected in a 1.5-mL tube, mixed with 100 µL of sterile distilled water, and vortexed for 60 s. The supernatant (1 µL) contained the template DNA for the LAMP reaction. In addition, the root samples were placed on NARM medium and cultivated for 1-3 days at 25 • C. The mycelia grown were transferred to V8 medium or slants for identification based on their morphological and molecular characteristics.

Identification of G. sylvaticum
In total, 156 root-disease samples from 38 plant types were collected from 17 counties in seven cities in Guizhou. The samples were cultured in a selective medium, and 161 isolates of Globisporangium were obtained. All isolates were initially identified via ITS sequencing, of which 29 showed 99.5-100% similarity to G. sylvaticum. Furthermore, 11 of the 29 isolates were obtained from different plants ( Table 2) and further identified using the cox1 sequence. A phylogenetic tree of the two sequences was constructed. We observed that the 11 isolates exhibited minimal association with the phylogenetic cluster containing G. sylvaticum CBS 543.67, G. sylvaticum BR647, G. terrestre (P. terrestris) CBS 112,352, and G. terrestre BR922 ( Figure 1, Table 1).   Furthermore, all 11 isolates were heterothallic and were grown in cultures paired with opposite mating-type isolates before morphological identification. The morphologies of GZsh01 × GZco02 on V8A are shown in Figure 2. Smooth, terminal, or intercalary oogonia were observed with a diameter of 19-20.5 µm (average: 20 µm). Approximately 2-4 antheridia were present per oogonium, which were diclinous. The aplerotic oospores had a diameter of 15-18 µm (average: 16.5 µm) and a wall with a thickness of 1-2 µm. We observed increased hyphal swelling, which was globose, limoniform, intercalary, or terminal ( Figure 2). The observed morphology was similar to that of G. sylvaticum CBS 234.68×230.68. Therefore, we finally identified the isolated strain as G. sylvaticum.

Host Plants of G. sylvaticum in Guizhou, China
According to reports, G. sylvaticum can infect more than ten different crops, including apples, carrots, lettuce, and soybeans [34][35][36]. The infection of pumpkin, eggplant, spring onions, and green beans by G. sylvaticum has not been previously reported; however, the infection of these plants was identified from the plant samples used in this study. A pathogenicity test was performed to determine whether these four plants were hosts of this pathogen. Figure 3 shows the growth results of the inoculated plants, which revealed that among the infected plants, pumpkin, spring onion, and green bean had poor development, and eggplant had slightly poor growth with a decrease in the number of roots and exhibited browning symptoms. Plants in the control group remained symptom-free. The pathogen was reisolated from the diseased roots of all inoculated plants and confirmed to be G. sylvaticum.

LAMP and PCR Primer Design and Specificity
Primer sets targeting the ITS region were designed according to the principles of species-specific LAMP and PCR primer design. In total, six LAMP primer sets were designed, each consisting of a modified set on either the F side (F3, FIP) or the B side (B3, BIP) of the primer set. These individual sets were then combined to enhance specificity, and one additional PCR primer set was designed separately.
All primer sets were screened using DNA extracts from the eight isolates in Globisporangium. The modified set was selected for LAMP because it produced the most specific and consistent amplification results ( Figure S1). Two loop primers were designed and added to the selected set ( Figure S1). The specificity tests were repeated ( Figure 4A,B). PCR primers were also tested ( Figure 4C). In addition, the primer sets (Table 3) were further tested with a wide variety of Globisporangium and other species and were shown to be highly specific for G. sylvaticum (Table 2). Furthermore, a temperature gradient from 60 to 67.5 • C was used to optimize the LAMP reactions for this primer set ( Figure S2). An optimal temperature of 65 • C where the reactions exhibited excellent specificity, efficiency, and stability was selected.

Sensitivity of LAMP or PCR In Vitro
Serial dilutions of G. sylvaticum (GZst02) genomic DNA were used to evaluate the detection limit of the LAMP method using the selected primer set at the optimal temperature. The sensitivity of the LAMP primers was 1 pg/μL based on real-time fluorescence

Sensitivity of LAMP or PCR In Vitro
Serial dilutions of G. sylvaticum (GZst02) genomic DNA were used to evaluate the detection limit of the LAMP method using the selected primer set at the optimal temperature. The sensitivity of the LAMP primers was 1 pg/µL based on real-time fluorescence intensity and SYBR Green I dye analysis, as illustrated in Figure 5A,B. The same dilutions were used for PCR, demonstrating a sensitivity of 10 pg/µL ( Figure 5C). intensity and SYBR Green I dye analysis, as illustrated in Figures 5A,B. The same dilutions were used for PCR, demonstrating a sensitivity of 10 pg/μL ( Figure 5C).

Detection of the Pathogen in Field Samples
In total, 45 diseased root samples were obtained from nine plant types (5 of each) in Guizhou ( Figure 6). The pathogen was detected using the P-LAMP assay with SYBR Green I dye; brown indicated a negative result, whereas green showed a positive result. Globisporangium sylvaticum was found in 29 samples, including 5 eggplant, 4 cucumber, 5 pumpkin, 5 green bean, 3 spring onion, 2 corn, and 5 cabbage samples. The pathogen was not detected in lettuce and rice samples ( Figure 6). The isolates were recovered on NARM agar from all positive samples and were confirmed to contain G. sylvaticum based on taxonomic characteristics.

Detection of the Pathogen in Field Samples
In total, 45 diseased root samples were obtained from nine plant types (5 of each) in Guizhou (Figure 6). The pathogen was detected using the P-LAMP assay with SYBR Green I dye; brown indicated a negative result, whereas green showed a positive result. Globisporangium sylvaticum was found in 29 samples, including 5 eggplant, 4 cucumber, 5 pumpkin, 5 green bean, 3 spring onion, 2 corn, and 5 cabbage samples. The pathogen was not detected in lettuce and rice samples ( Figure 6). The isolates were recovered on NARM agar from all positive samples and were confirmed to contain G. sylvaticum based on taxonomic characteristics.

Detection of the Pathogen in Field Samples
In total, 45 diseased root samples were obtained from nine plant types (5 of each) in Guizhou ( Figure 6). The pathogen was detected using the P-LAMP assay with SYBR Green I dye; brown indicated a negative result, whereas green showed a positive result. Globisporangium sylvaticum was found in 29 samples, including 5 eggplant, 4 cucumber, 5 pumpkin, 5 green bean, 3 spring onion, 2 corn, and 5 cabbage samples. The pathogen was not detected in lettuce and rice samples ( Figure 6). The isolates were recovered on NARM agar from all positive samples and were confirmed to contain G. sylvaticum based on taxonomic characteristics.

Discussion
We collected several root-diseased samples from important cash crops in different parts of Guizhou, and a total of 13 Globisporangium species were found, indicating the substantial distribution potential of Globisporangium in Guizhou. Therefore, it is crucial to study prevention and control strategies for crop diseases caused by Globisporangium. Globisporangium sylvaticum exhibited the highest isolation frequency among the obtained species, indicating that it was the dominating species in the region. Host range and rapid detection methods of this pathogen were thoroughly explored in this study to assist in preventing the pathological development of this pathogen and of the pathogenic Globisporangium genus.
Eleven suspected isolates from various plants were identified using molecular and morphological methods. According to the constructed phylogenetic tree, there was no difference in the clustering distance between the isolates and G. sylvaticum or G. terrestre; furthermore, some sequence differences were found between them, indicating that these pathogens are likely to have relatively rich genetic diversity. Our isolates were eventually identified as G. sylvaticum mainly based on heterothallic and morphological characteristics. Globisporangium terrestre was first reported by Paul (2002), and the ITS sequence was deposited in GenBank (accession number AY039714) [37]. However, we further "Blast" this sequence and found that it is very different from the now known G. terrestre CBS112352 (HQ643857). Robideau et al. (2011) showed that the ITS sequence could not, while cox1 could, distinguish G. terrestre from G. sylvaticum in molecular systematics, but both remained in the same cluster [32]. In addition, G. terrestre is considered to be homothallic, in contrast to G. sylvaticum, but the sexual structures produced by both are similar. Therefore, all strains of these species, including G. sylvaticum isolated from Guizhou, demonstrated the need for further taxonomic study.
We discovered various mating types, such as GZsh01 × GZco02, in the region, suggesting that the initial infection sources of the pathogen were probably overwintering oospores.
Based on the pathogen analysis or Koch's postulates between G. sylvaticum and the isolated crops, the pathogen is currently known to have eleven hosts, including four novel hosts identified in Guizhou. This vast host range is consistent with the widespread range of hosts that Globisporangium is known to inhabit commonly [38]. The results of the pathogenicity experiment showed that the pathogenic ability of the same strain differed among various hosts. G. sylvaticum exhibits no host-specificity [39]. However, further studies are required to prove this feature.
Additionally, we investigated the potential of LAMP to detect G. sylvaticum using simple DNA extraction. A LAMP primer set including six primers was designed for this pathogen and was confirmed to be specific. Unfortunately, based on the sequence comparison, the primer set was unable to distinguish G. terrestre. Globisporangium terrestre has only been found in soil as well as soybeans [40]. As a result, the LAMP primers designed here are still practical for G. sylvaticum in most host plants. However, highly specific G. sylvaticum primers need to be further developed. The reaction was also sensitive, with an acceptable detection limit of 1 pg/µL, which is higher than that of PCR (10 pg/µL). The reaction process was easy to perform and only took 1 h, and the amplification results were successfully measured based on visual observation with colorimetric indicators or real-time fluorescence using a LAMP fluorescent dye. The LAMP products can easily form aerosols, resulting in contamination that is difficult to remove [41]. Thus, to avoid contamination, we added mineral oil to overlay the reaction mixture. Due to the mineral oil overlay, LAMP-amplified products were not recovered for further testing, and electrophoresis was not performed. Furthermore, many researchers have reported that colour change results were consistent with electrophoresis results [42,43]. Therefore, LAMP is an alternative nucleotide amplification method that is rapid, simple, highly sensitive, and suitable for analysing pathogens in the field.
Temperature is the most important factor for a successful reaction and amplification because the pairing ability of primers and template DNA and the efficiency of the amplification enzyme are closely related to temperature. A temperature range of 60-67.5 • C was tested here for optimisation of the reaction. The results showed that G. sylvaticum can be specifically and rapidly detected at 65 • C. A higher temperature may increase the specificity of the reaction but reduce the amplification efficiency [44]. To accelerate the amplification, a pair of loop primers was designed and added into the reaction mixture, and the reaction was amplified within 30 min. Although loop primers are not typical primers for LAMP amplification, their addition can significantly improve the reaction rate and shorten reaction time [45].
For the on-site detection of G. sylvaticum in the field, we employed the P-LAMP approach. This method employs a crude DNA extraction method that does not require any reagent processing, takes only 1-2 min, and is considered highly efficient for detecting G. irregulare or Phytophthora colocasiae from plant roots or taro leaves [33,46]. Here, we performed P-LAMP on root samples of 45 plants from nine species and identified the isolates from the tissue cultivated on the NARM medium to verify the detection results. These results demonstrated that the P-LAMP-positive samples contained G. sylvaticum. Negative results were observed in 16 samples, particularly in lettuce and rice. These results may be attributed to the potential infection of the symptomatic seedlings by fungi or other oomycetes [47][48][49] or the possibility of inadequate detection of root samples. The former suggests the necessity of implementing a comprehensive plant disease diagnosis system that effectively encompasses all major pathogens of the plant. The latter emphasizes the need to conduct multiple tests on negative samples in order to ensure full verification. In summary, P-LAMP can be used to efficiently detect G. sylvaticum in plant roots because of its simplicity, reliability, rapidity, and low cost.

Conclusions
In this study, we investigated the distribution of Globisporangium, especially G. sylvaticum, among important cash crops in various regions of Guizhou. Isolates were identified, and their hosts were analysed. G. sylvaticum is common in all regions of the Guizhou and has eleven hosts, including four novel hosts identified here. Hence, further research on the diversity of the pathogenic Globisporangium genera in Guizhou would have important scientific value. In addition, we designed a LAMP primer set with a specificity and sensitivity of 1 pg/µL to detect G. sylvaticum and demonstrated that P-LAMP has the potential to detect pathogens in agricultural fields and may provide a significant contribution to management and prevention, even during the early onset of disease in the field.