Occurrence of Eutypella species on cotton (Gossypium hirsutum L.) in New South Wales, Australia

We report for the first time the occurrence of an Eutypella species on cotton in New South Wales (NSW), Australia. The putative Eutypella was isolated from dead cotton plants that exhibited atypical symptoms of either Fusarium or Verticillium wilt. Dead plants had symptoms resembling lightning strike damage with blackened outer bark around the fifth node zone. Internal vascular discoloration was reddish grey and often occurred in V-shaped sections. Sequences of the internal transcribed spacer (ITS) revealed a 93.7–94.1% similarity to Eutypella scoparia, indicating that the NSW cotton isolates represented a putatively novel species within the Eutypella genus. Pathogenicity of the isolated Eutypella was assessed on cotton stem cuts and living plants inoculated with culture agar plugs. Typical black necroses on outer bark and reddish vascular discoloration were reinduced in both Eutypella-inoculated stem cuts and living plants. Subsequently, the same Eutypella pathogen was successfully recovered, thus fulfilling Koch’s postulates. The detection of Eutypella on NSW cotton is believed to be an independent event from a recently reported occurrence of Eutypella on Queensland cotton. The Queensland detection occurred in the same 2017/18 cropping season at approximately 730 km apart and was associated with distinct Eutypella isolates.


Introduction
Cotton (Gossypium hirsutum L.) is a cash crop for regional New South Wales (NSW) and Queensland (QLD), Australia. The annual generated revenue is approximately AUD 1.9 billion (CRDC 2018). The sustainable production of Australian cotton relies on successful management of abiotic stress such as drought as well as biotic stresses i.e. pests and diseases (Otto 2020). Of these stresses, diseases caused by fungal pathogens such as Alternaria alternata (Le and Gregson 2019), Thielaviopsis basicola (recently described as Berkeleyomyces spp. by Nel et al. (2018)) (Nehl et al. 2004), Fusarium oxysporum f. sp. vasinfectum (Kirby et al. Duy Phu Le duy.le@dpi.nsw.gov.au;lephuduy08@gmail.com which was atypical to those of Verticillium wilt and Fusarium wilt in cotton, was detected upon cutting at the ground level. The vascular discoloration was more profound at the blackened bark zone and downward to the below ground parts but did not appear systemic upward beyond the blackened zone (Fig. 2B). The discoloration of the tissue was typically reddish grey (Fig. 2C), and often occurred in wedges (Fig. 2D). These symptoms allowed for differentiation of these unusual dead plants from others affected by Verticillium and Fusarium wilt diseases.
In Queensland, Smith et al. (2022) reported an undescribed Eutypella species that was associated with death of cotton plants that exhibited the similar symptoms to those observed in NSW. The report was based on a detection event in central QLD in 2017/18 cropping season. Independently, we also first noticed the unusual death of cotton plants in northern NSW in 2017/18 season. Therefore, this study aimed to identify if the two independent detection events of these unidentified dead plants in such a geographic distance (approximately 730 km apart by road) was associated with the same causative fungal pathogen.

Pathogen isolation
In NSW, dead plants were sampled and brought back to the laboratory for putative pathogen isolation and identification. The isolation method from cotton stems was adopted from Le et al. (2020b). The isolation was initiated by excising small sections with vascular discoloration, 1-2 cm long from the stems and peeling off the outer bark, followed by surface decontamination for 30 s with 70% ethanol. Then, the sections were blotted dry with paper towel and discolored wood chips were thinly excised using a sterile scalpel and embedded into potato dextrose agar (PDA Difco) amended with 100 ppm streptomycin sulfate (Sigma Aldrich) (sPDA) in Petri plates. The plates were sealed with parafilm and incubated at 25 °C in the dark for 3-5 days. Putative fungal colonies that emerged from vascular tissues were individually sub-cultured onto new sPDA plates and single hyphal cultures established. Pure cultures were stored in sterile water at room temperature for subsequent experimentation.  A dead cotton plant exhibiting symptoms of lightning strike damage that being blackened necrosis of the outer bark which was consistently observed at around the fifth node zone, noting all leaves remained strongly attached to the stems (A); internal vascular discoloration was observed upon removal of the black necrotic bark and the discoloration did not appear far beyond the black necrotic area (B); typical profound reddish grey vascular discoloration was noticed on lower stem and tap root (C); wedge-shaped discoloration often observed on a cross-cut section (D) electrophoresis. The products were run at 100 V in a 1.5% agarose gel for 45 min, pre-stained with GelRed® Nucleic Acid Gel Stain (Biotium) and visualised under UV light using a UVIDOC HD6 (UVITEC Cambridge). All PCR products were purified and sequenced by Macrogen Inc., South Korea.

Assay 1
Pathogenicity of the recovered fungus was assessed on cotton stem sections. Cotton (cv. Sicot 75RRF, relatively mild resistance to wilt diseases caused by Fusarium oxysporum f. sp. vasinfectum and Verticillium dahliae) was grown and maintained in a temperature-controlled glasshouse until fruiting. Unless otherwise stated, cultivar Sicot 75RRF was used in pathogenicity assays and all plants were grown from black cotton seeds. Healthy looking stems were selected and excised into approximately 30 cm long sticks, and surface decontaminated twice with 70% ethanol for 1 min. After blotting dry with paper towel, a small Sect. (0.5 × 1 cm) of the outer bark was carefully flipped up using a sterile surgical scalpel. Plugs (0.5 × 0.5 cm) of active growing fungal cultures on sPDA were excised and placed upside down onto the opened flipped sections. The flipped bark was gently set back in place and sealed with Parafilm. There were three representative isolates (Table 1) and six sticks per isolate. Once inoculated, stems were bulked and bagged in plastic zip-lock bags according to isolate. The bags were moistened with sterile water, sealed tight and kept in the dark at 25 °C for two weeks before visual assessments for external and internal discoloration were undertaken. The control stems were treated in the same manner using sterile sPDA plugs for inoculation. The assay was conducted twice.

Pathogen identification
Genomic DNA extraction was conducted using a Wizard ® Genomic DNA Purification Kit (Promega, Sydney Australia) with slight modifications adopted from Le et al. (2020b). Briefly, 10-100 mg of mycelia were scraped off culture plates and transferred into a 1.5 mL tight-lock Eppendorf tube. The mycelia were macerated and 500 µL Nuclei Lysis Solution added, then incubated at 65 °C for 30 min. Subsequently, a volume of 3 µL RNase A Solution was added to each of the tubes and followed by another incubation at 37 °C for 15 min. Once the solution returned to ambient temperature, 200 µL of Protein Precipitation Solution was added and vortexed vigorously to homogenicity, followed by a centrifugation at 13,000 rpm for 5 min. The supernatants were carefully pipetted to new 1.5 mL Eppendorf tubes containing 600 µL room-temperature isopropanol. The tubes were gently inverted and centrifuged at 13,000 rpm for 5 min. The supernatants were carefully decanted, and the visible DNA pellets were washed twice with 70% roomtemperature ethanol. The DNA pellets were air-dried under a fume hood for 30-45 min, rehydrated with 50-200 µL DNA Rehydration Solution depending on the size of the pellets and incubated at 65 °C for 1 h. Finally, the DNA solutions were stored at − 20 °C.
PCR amplification of the internal transcribed spacer (ITS) was conducted with a primer pair of ITS4 5'-TCCTCCGCT-TATTGATATGC-3' and ITS5 5'-GGAAGTAAAAGTC-GTAACAAGG-3' (White et al. 1990) using GoTaq® G2 Green Master Mix (Promega). Each PCR mix contained: 10 µL of Green Master Mix, 8 µL of DNase free water, 1 µL of 10 mM primer mix and 1 µL of DNA template. DNase free water was included as a negative (no-template) control. PCR cycling conditions were adopted from Le and Gregson (2019) as follows: initial denaturation for 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at 52 °C and 90 s at 72 °C, with a final elongation step of 7 min at 72 °C. Integrity of amplified PCR products was confirmed by The means of necrotic length (mm) data were bulked from the two pathogenicity assays due to a similar level of symptom expression. Means followed by the same letter were not significantly different (p = 0.05) 2 Percentage of ITS sequence similarity was extracted from BLASTN search. GenBank accession number for E. scoparia was MK336539.
3 A representative isolate CQ9-3a from the QLD collection was selected for a pairwise comparison since it was well characterised (Smith et al.2022). ITS sequence of CQ9-3a was deposited in GenBank as MZ918892.
Eutypella scoparia (GenBank accession No. MK336539) with the percentage of identity of 93.7-94.1 (Table 1). Additionally, the ITS sequences of the three representative NSW isolates only shared 89.1-89.3% similarity in comparison to a recently reported Eutypella isolate (CQ9-3a) that was recovered from cotton in QLD (Smith et al.2022) (Table 1). In a maximum likelihood analysis, the three NSW Eutypella isolates were well separated from the reference Eutypella and related species in the Diatrypaceae and the QLD isolates (Fig. 3). This indicated that the NSW cotton Eutypella isolates represented a putatively novel species. However, we only report here the first occurrence and identification of undescribed Eutypella species on cotton in NSW due to the lack of morphological data.

Assay 1
In the repeated assays on the stem cuts, the three tested isolates were able to infect the stems and induce external as well as internal symptoms that were similar to those observed in the field. External black necroses were obvious around the inoculation point a week after inoculation at 25 °C; and the black necroses continued to progress and were more profound at two weeks after inoculation (Fig. 4A). The mean external necrotic length varied among isolates from 7.5 to 16.1 mm (Table 1). Similarly, internal vascular discoloration was only observed on Eutypella-inoculated stems (Fig. 4B). The mean length of internal vascular discoloration ranged from 19.1 to 21.5 mm ( Table 1). The same pathogen was fully recovered from infected stems and identified using the ITS sequences, thus fulfilling Koch's postulates.

Assay 2
Initial black necroses were noticeable around Eutypellainoculated points at six weeks after inoculation. The lesions continued to enlarge with time. No obvious foliar symptoms were detected on both Eutypella-inoculated and control plants. At eight weeks after inoculation, some infected plants started wilting. These plants were considered dead once the wilting was irreversible. Once dead, leaves were dried out and remained green and attached strongly to the plants, as observed in the field. Upon termination and removal of cotton wool coverings, external black necroses were only observed on the Eutypella-inoculated plants. Mean external necrotic length varied from 35.5 to 59.5 mm (P = 0.77) (Fig. 5graph). Meanwhile, stem gall formations at inoculated points were only noticed on the control plants (Fig. 5A). A cross stem cut revealed a reddish vascular discoloration (Fig. 5B), which was characteristic for Eutypella

Assay 2
Three-month-old cotton plants (grown individually in 250 mm dia. pots) were stem-inoculated with actively growing Eutypella plugs. Inoculation was performed as described in assay 1. To protect the inoculation sites from drying out, sterile moistened cotton wool was placed over the top of the inoculation sites and sealed with parafilm. Control plants were inoculated with sterile sPDA plugs. There were four replicate pots per isolate. The trial was carried out in a temperature-controlled glasshouse (25-30 °C). All pots were watered daily and monitored for symptom expression for two months.

Assay 3
Fifteen cotton seeds per pot were sown in 140 mm dia. pots containing pasteurised Searles® potting mix. Unless otherwise stated, the pasteurised Searles® potting mix was used for all pot trials. At four weeks after sowing, cotton seedlings were thinned down to 10 seedlings per pots. All 10 seedlings, along with potting mix, were gently removed from the 140 mm dia. pot. The lower half of the root system and potting mix were trimmed off to simulate open wound damage and transferred into 250 mm dia. pots containing Eutypella-infested potting mix. Two-week-old actively growing Eutypella cultures on sPDA plates (90 mm dia.) were excised into 5 mm plugs and incorporated into the potting mix. There were five replicate pots per isolates and one plate per pot. Control plants were treated in the same manner; however, the potting mix was only incorporated with clean uncolonized sPDA plate. Then, all pots were randomised and maintained in a temperature-controlled glasshouse (25-30 °C). All pots were watered daily and monitored for symptom expression for the next three months.

Isolation and identification
A total of 10 pure fungal isolates was obtained from dead cotton plants that exhibited atypical symptoms caused by Fusarium oxysporum f. sp. vasinfectum and Verticillium dahliae. Cotton-like white cultures appeared slightly aerial on sPDA at first and turned dark with age. No reproductive structures of the cultures were observed on sPDA cultures at different ages. Therefore, the identification was solely based on the DNA sequences of the ITS region. The ITS sequences (512 bp) of three representative isolates were obtained for pathogen identification. The closest sequence similarity of the three putative Eutypella isolates was to control plants. Subsequently, no Eutypella was recovered from either Eutypella-inoculated or control plants.

Discussion
This study reports for the first time the occurrence of Eutypella species on cotton in NSW. The occurrence of Eutypella species on cotton in NSW was deemed independent from the QLD detection (Smith et al. 2022) since it was detected on two different cultivars (cv. Sicot 746B3F in NSW vs. Sicot 714B3F in QLD) at a similar time in field-infected plants. Then mean length of internal vascular discoloration ranged from 61.3 to 136.3 mm (P = 0.69). The same Eutypella was fully recovered and identified.

Assay 3
Three months after inoculation, there was neither obvious foliar nor stem canker symptoms observed on the Eutypellainoculated or control plants. Upon termination of the assay due to a heavy infestation with whiteflies and two spotted mites, no vascular discoloration was observed either on tap roots or main stems of both Eutypella-inoculated and Fig. 3 Maximum likelihood tree constructed from the ITS sequences showing a relative relationship among undescribed Eutypella isolates that were recovered from cotton grown in QLD and NSW and other closely related species in the Diatrypaceae family. Bootstrap frequencies from 1,000 replications are shown next to the branches. Only values higher than 50 are shown the 2017/18 cropping season. Additionally, the recovered Eutypella isolates from NSW and QLD were highly distinct. Firstly, a representative isolate (CQ9-3a) from QLD readily produced an abundance of filiform conidia, while none of the three NSW representative isolates sporulated on sPDA. Secondly, the maximum likelihood analysis of the ITS sequences revealed a relatively distant group of NSW isolates to the QLD isolates. Several species descriptions within the Eutypella genus were only based on ascospore morphology and DNA sequences (Mehrabi et al. 2019;Trouillas et al. 2011). However, ascospores from cotton stubble has not been discovered to date. Therefore, a report of an undescribed species of Eutypella on cotton would allow for the confirmation of the occurrence of a novel pathogen on cotton.
We are also able to demonstrate a direct association of this undescribed NSW-Eutypella species with unusual death of cotton plants. In stem cut assays, the three tested NSW-Eutypella isolates were able to induce external black necrosis only a week after inoculation, which was identical to field symptoms. Subsequent recovery of the same pathogen from the inoculated stem cuts allowed for the confirmation of its pathogenicity. Similarly, using the same inoculation method on living cotton plants, two out of the three isolates were able to cause plant death at two months after the inoculation. Smith et al. (2022) used a similar stem inoculation approach with agar plugs and was able to confirm the pathogenicity of their QLD-Eutypella representative isolate. However, the direct association of the QLD-Eutypella isolate and dead cotton plants has not been elucidated. This Funding Open Access funding enabled and organized by CAUL and its Member Institutions Open Access funding enabled and organized by CAUL and its Member Institutions

Data Availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations
Competing Interests The authors declare that they have no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. could be due a different level of aggressiveness between the NSW-and QLD-Eutypella isolates. Additionally, it could be associated with a different level of host responses since we used two different cultivars in our pathogenicity assays. Therefore, it will warrant additional research to assess the virulence of these undescribed Eutypella species against the susceptibility of Australian commercial cultivars.
Unlike Eutypa lata, the causal agent of Eutypa dieback of grapevine, epidemiology of Eutypella species in association with tree trunk disease has not been well-elucidated (Úrbez-Torres et al. 2020). On cotton, species of Eutypella were considered to be a novel pathogen on this host crop (Smith et al.2022); therefore, knowledge about the disease epidemiology and pathogen biology is limited. Smith et al. (2022) were uncertain about the soilborne infection pathway of QLD-Eutypella though they successfully manipulated the occurrence of a dead cotton plant using field soil collected from a Eutypella-infested site. We failed to induce Eutypella infection on cotton plants growing in potting mix inoculated with mycelial mass, even when the root was heavily trimmed. Similarly, Smith et al. (2022) also commented that they were not successful in inoculating healthy cotton from the root using mycelial inoculants. Mycelia were commonly used to assess the pathogenicity of Diatrypaceae species, including Eutypella (Moyo et al. 2018;Úrbez-Torres et al. 2020). However, the infective capacity of mycelia was not as consistent as ascospores, which are the primary infectious and dispersing structures (E.S. Scott, The University of Adelaide, personal communication). Therefore, it is warranted to reassess the soilborne infection pathway of Eutypella on cotton using ascospore inoculants once the ascospore is discovered.
The genus Eutypella belongs to the Diatrypaceae family which occur worldwide on woody crops and forest trees (Úrbez-Torres et al. 2009;Trouillas et al. 2011;Brglez et al. 2020). In Australia, Eutypella species such as E. australiensis, E. citricola, E. cryptovalsoide and E. microtheca have been recorded on grapevine, citrus, ficus and acacia; however, the pathogenicity status of these remained unknown (Trouillas et al. 2011). Other species such as E. parasitica and E. vitis were identified as pathogens of concern on maple trees and grapevines in the USA and Europe, respectively (Davidson and Lorenz 1938;Úrbez-Torres et al. 2009;Brglez et al. 2020 Smith et al. (2022), we can confirm the pathogenicity of newly recovered Eutypella isolates and its lethal infection to cotton in NSW.