Neopestalotiopsis asiatica strain CP1 Causing Leaf Blight on Zedoary Turmeric in Guangxi, China

doi:10.21203/rs.3.rs-4190389/v1

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Neopestalotiopsis asiatica strain CP1 Causing Leaf Blight on Zedoary Turmeric in Guangxi, China

https://doi.org/10.21203/rs.3.rs-4190389/v1

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Background

Zedoary turmeric (Curcuma phaeocaulis), a cornerstone of traditional Chinese medicines in Guangxi Province, holds immense significance. Regrettably, the emergence of a novel leaf wilt in October 2017 has cast a pall over its production, leading to detrimental impacts on both yield and quality.

Results

By meticulous single-conidial isolation, this new pathogen was successfully extracted from ten diseased leaves. Subsequent confirmation of pathogenicity was achieved via the meticulous execution of Koch's postulates. In pursuit of accurate identification, morphological data were harmonized with a concatenated sequence analysis of the rDNA internal transcribed spacer (ITS), translation elongation factor 1 (TEF), and β-tubulin (TUB) regions. This rigorous approach unveiled the pathogen as an Neopestalotiopsis asiatica, warranting the nomenclature Neopestalotiopsis asiatica strain CP.

Conclusions

This is the first description on Neopestalotiopsis asiatica as causal agent for leaf blight on zedoary turmeric. The ramifications of this pathogen's definitive identification and ongoing surveillance cannot be overstated, signifying a pivotal alert to zedoary turmeric cultivators in confronting this newfound peril.

Chinese herbal medicine

Fungi

Morphology

Phylogeny

Zedoary turmeric (Curcuma phaeocaulis), a member of the family Zingiberaceae, has been cultivated for medicinal purposes in China for an extensive duration (Chen et al. 2019). Primarily grown in southern provinces such as Sichuan, Fujian, Zhejiang, and Guangxi, this plant has historically been employed to ameliorate conditions such as blood blockage, dysmenorrhea, and trauma (Ni et al. 2021). In addition, its volatile oils, curcumin analogs, and trace elements are recognized for their substantial medicinal attributes, encompassing anti-inflammatory (Hou et al. 2015), antibacterial, antiviral, antitumor, and antipathogen effects (Shi et al. 2021).

Zedoary turmeric typically undergoes individual cultivation, with one or a few commercial cultivars. Although relatively manageable, such cultivation practices render these cultivars susceptible to disease outbreaks. Leaf diseases, particularly, hinder the growth and development of zedoary turmeric, culminating in yield loss. Qinzhou City in Guangxi Province stands as China's foremost zedoary turmeric producer, boasting a planting area of 4,333 hectares and an economic value of 73 million U.S. dollars in 2015. However, since 2017, leaf blight has progressively intensified, resulting in over a 20% reduction in yield within Guangxi. The identity of the causative pathogen remains elusive, and the absence of scientific and efficacious control measures exacerbates the situation. The initial stages of the plant's leaf blight manifest as small yellowish spots on the leaves, gradually expanding towards their tips or margins. Subsequently, the spots propagate across the entire leaf, deepening in hue and dispersing minute black spots on the surface. These symptoms deviate from those induced by fungi such as Curvularia (Chen et al. 2013), Nigrospora sphaerica (Zhang et al. 2011), Colletotrichum Curcumae (Li et al. 2016) and Phoma matteucciicola (Zheng et al. 2018), suggesting a potential novelty in the fungal pathogen responsible.

Traditional fungal taxonomy relies on morphological characteristics, however, due to limited morphological indicators, it may inadequately reflect the evolutionary relationships between species. With the advancement of sequencing methodologies, DNA barcodes have emerged as a potent tool, utilizing standardized DNA segments like ITS (internal transcribed spacer), TUB (β-tubulin), TEF (translation elongation factor 1 α), ACT (actin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and CAL (calmodulin) genes for species recognition. This technique expedites precise identification based on nucleotide sequence differences. At present, the ITS gene serves as the primary barcode gene for fungal classification. However, multigene phylogenetic analysis has been shown to offer a more comprehensive depiction of interspecific fungal relationships. In 2014, Maharachchikumbura et al. (2014) employed a combination of ITS, TUB, and TEF gene sequences to classify Pestalotiopsis species, surpassing the ITS approach. Subsequent to generic reclassification, Pornsuriya et al. (2020) utilized the method proposed by Maharachchikumbura et al. to identify pathogens responsible for rubber tree defoliation within the Neopestalotiopsis group, classifying them as N. cubana and N. formicarum.

The purpose of this study was to identify the pathogenic fungi causing zedoary turmeric leaf blight in Guangxi. Morphological identification and molecular phylogenetic analysis were conducted to this purpose. Koch's postulates were applied to validate the pathogenicity of the isolated fungi against zedoary turmeric.

Field survey and fungal isolation

Zedoary turmeric (Curcuma phaeocaulis) leaves exhibiting blight symptoms were collected from production fields located in Nanxiong, Guilin city, Guangxi Province, during Oct 2017. The collected diseased leaf specimen (accession number: GXMG0039667) was subsequently preserved in the Guangxi Botanical Garden of Medicinal Plants. Samples were collected in the field on approximately 10 plants of Zedoary turmeric (Curcuma phaeocaulis), randomly selected, and were brought to the Plant Pathology laboratory of the College of Plant Protection at the Southwest University of Chongqing. One hundred small sections (4 mm × 4 mm) of the Leaves were immersed for 45 s in 75% ethanol, followed by 1% sodium hypochlorite (NaClO) for 3 min, rinsed in sterile distilled water, dried on sterile absorbent paper, and placed on water‒agar (WA) plates and incubated at 25℃, alternating between 12 h cycles of light and darkness generated by fluorescent lighting, over a span of 3 days. Transplanted hyphal margins were transplanted to potato dextrose agar (PDA) plates to foster conidial production, and the ensuing cultures were maintained at 25℃. Single-spore isolates were obtained from conidia produced in pure cultures grown on PDA.

Morphological characteristics

Circular mycelial discs, measuring 6 mm in diameter and sourced from the strains, were centered upon PDA medium. The cultures were incubated in continuous darkness for a duration of seven days, during which the attributes of the colonies were meticulously documented. Daily observations encompassed the recording of colony color as well as the diameters along two vertical axes. Subsequent to the colony observation phase, the isolates were subjected to additional incubation on PDA plates maintained at a temperature of 25℃ over a span of 21 days, promoting the generation of conidia. In a quest for comprehensive characterization, conidia lacking appendages underwent measurement for their length and width, with an aggregate sample size of 100. Moreover, the length of the three median cells, along with the dimensions of both apical and basal appendages, were also gauged. The quantification of apical appendages was performed under the meticulous examination of an OLYMPUS BX53F microscope (Olympus, Tokyo).

DNA extraction, polymerase chain reaction (PCR) and sequencing

The genomic DNA was extracted from surface mycelium scraped off from pure cultures, the cetyltrimethylammonium bromide (CTAB) method (Tsuji et al. 2017). For nucleotide sequence comparisons, the first internal transcribed spacer region, the 5.8S nrRNA gene, the second internal transcribed spacer region and the 50 end of the 28S nrRNA gene (ITS), and the partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) genes were amplified using primer pairs ITS5/ITS4 (White et al., 1990), EF1-728F/EF-2 (O'Donnell et al. 1998; Carbone & Kohn 1999), and Bt2a/Bt2b (Glass & Donaldson 1995). Amplification conditions for ITS and TEF followed Crous et al. (2013) and for TUB, Lee et al. (2004). The resulted PCR products were purified, cloned and custom sequenced (Shanghai Shenggong Sequencing Company) using the same primer sets of the PCR reactions. The newly generated sequences were aligned to a representative matrix of Neopestalotiopsis, selecting two species of Pestalotiopsis as an outgroup. The GenBank accession numbers of sequences used in these analyses are given in Table 1.

Table 1

Information of fungal isolates used in the phylogenetic analysis and their corresponding GenBank accession numbers. Isolates in bold are from this study.
Species	Strain¹	Host/Substrate	Origin	GenBank accession number²
Species	Strain¹	Host/Substrate	Origin	ITS	TEF	TUB
Neopestalotiopsis acrostichi	MFLUCC 17-1754 T	Acrostichum aureum	Thailand	MK764272	MK764316	MK764338
N. alpapicalis	MFLUCC 17-2544 T	Rhizophora mucronata	Thailand	MK357772	MK463547	MK463545
N. aotearoa	CBS 367.54 T	Canvas	New Zealand	KM199369	KM199526	KM199454
N. asiatica	MFLUCC 12–0286 T	Unidentified tree	China	JX398983	JX399049	JX399018
N. asiatica	strain P817	Almond	Iran	KM074046	KM074052	KM074055
N. asiatica	strain CP1	Zedoary turmeric	China	ON406217	ON409190	ON409191
N. australis	CBS 114159 T	Telopea sp.	Australia	KM199348	KM199537	KM199432
N. brachiata	MFLUCC 17-1555 T	Rhizophora apiculata	Thailand	MK764274	MK764318	MK764340
N. brasiliensis	COAD 2166 T	Psidium guajava	Brazil	MG686469	MG692402	MG692400
N. camelliae-oleiferae	CSUFTCC81 T	Camellia oleifera	China	OK493585	OK507955	OK562360
N. cavernicola	KUMCC 20–0269 T	Cave rock surface	China	MW545802	MW550735	MW557596
N. chiangmaiensis	MFLUCC 18–0113 T	Dead leaves	Thailand	N/A	MH388404	MH412725
N. chrysea	MFLUCC 12–0261 T	Pandanus sp.	China	JX398985	JX399051	JX399020
N. clavispora	MFLUCC 12–0281 T	Magnolia sp.	China	JX398979	JX399045	JX399014
N. coffeae-arabicae	HGUP 4019 T	Coffea arabica	China	KF412649	KF412646	KF412643
N. cubana	CBS 600.96 T	Leaf litter	Cuba	KM199347	KM199521	KM199438
N. drenthii	BRIP 72264a T	Macadamia integrifolia	Australia	MZ303787	MZ344172	MZ312680
N. egyptiaca	CBS 140162 T	Mangifera indica	Egypt	KP943747	KP943748	KP943746
N. ellipsospora	MFLUCC 12–0283 T	Dead plant materials	China	JX398980	JX399047	JX399016
N. eucalypticola	CBS 264.37 T	Eucalyptus globulus	N/A	KM199376	KM199551	KM199431
N. eucalyptorum	CBS 147684 T	Eucalyptus globulus	Portugal	MW794108	MW805397	MW802841
N. foedans	CGMCC 3.9123 T	Mangrove plant	China	JX398987	JX399053	JX399022
N. formicidarum	CBS 362.72 T	Dead Formicidae (ant)	Ghana	KM199358	KM199517	KM199455
N. guajavae	FMBCC 11.1 T	Psidium guajava	Pakistan	MF783085	MH460868	MH460871
N. guajavicola	FMBCC 11.4 T	Psidium guajava	Pakistan	MH209245	MH460870	MH460873
N. hadrolaeliae	COAD 2637 T	Hadrolaelia jongheana	Brazil	MK454709	MK465122	MK465120
N. hispanica	CBS 147686 T	Eucalyptus globulus	Portugal	MW794107	MW805399	MW802840
N. honoluluana	CBS 114495 T	Telopea sp.	USA	KM199364	KM199548	KM199457
N. hydeana	MFLUCC 20–0132 T	Artocarpus heterophyllus	Thailand	MW266069	MW251129	MW251119
N. iberica	CBS 147688 T	Eucalyptus globulus	Portugal	MW794111	MW805402	MW802844
N. iranensis	CBS 137768 T	Fragaria × ananassa	Iran	KM074048	KM074051	KM074057
N. javaensis	CBS 257.31 T	Cocos nucifera	Indonesia	KM199357	KM199548	KM199457
N. longiappendiculata	CBS 147690 T	Eucalyptus globulus	Portugal	MW794112	MW805404	MW802845
N. lusitanica	CBS 147692 T	Eucalyptus globulus	Portugal	MW794110	MW805406	MW802843
N. maddoxii	BRIP 72266a T	Macadamia integrifolia	Australia	MZ303782	MZ344167	MZ312675
N. magna	MFLUCC 12–0652 T	Pteridium sp.	France	KF582795	KF582791	KF582793
N. mesopotamica	CBS 336.86 T	Pinus brutia	Turkey	KM199362	KM199555	KM199441
N. musae	MFLUCC 15–0776 T	Musa sp.	Thailand	NR_156311	KX789685	KX789686
N. natalensis	CBS 138.41 T	Acacia mollissima	South Africa	NR_156288	KM199552	KM199466
N. nebuloides	BRIP 66617 T	Sporobolus jacquemontii	Australia	MK966338	MK977633	MK977632
N. olumideae	BRIP 72273a T	Macadamia integrifolia	Australia	MZ303790	MZ344175	MZ312683
N. pandanicola	KUMCC 17–0175 T	Pandanus sp.	China	N/A	MH388389	MH412720
N. pernambucana	URM 7148-01 T	Vismia guianensis	Brazil	KJ792466	KU306739	N/A
N. perukae	FMBCC 11.3 T	Psidium guajava	Pakistan	MH209077	MH523647	MH460876
N. petila	MFLUCC 17-1738 T	Rhizophora apiculata	Thailand	MK764276	MK764320	MK764342
N. phangngaensis	MFLUCC 18–0119 T	Pandanus sp.	Thailand	MH388354	MH388390	MH412721
N. piceana	CBS 394.48 T	Picea sp.	UK	KM199368	KM199527	KM199453
N. protearum	CBS 114178 T	Leucospermum cuneiforme	Zimbabwe	JN712498	KM199542	KM199463
N. psidii	FMBCC 11.2 T	Psidium guajava	Pakistan	MF783082	MH460874	MH477870
N. rhapidis	GUCC 21501 T	Rhododendron simsii	China	MW931620	MW980442	MW980441
N. rhizophorae	MFLUCC 17-1551 T	Rhizophora mucronata	Thailand	MK764277	MK764321	MK764343
N. rhododendri	GUCC 21504 T	Rhododendron simsii	China	MW979577	MW980444	MW980443
N. rosae	CBS 101057 T	Rosa sp.	New Zealand	KM199359	KM199523	KM199429
N. rosicola	CFCC 51992 T	Rosa chinensis	China	KY885239	KY885243	KY885245
N. samarangensis	MFLUCC 12–0233 T	Syzygium samarangense	Thailand	JQ968609	JQ968611	JQ968610
N. saprophytica	MFLUCC 12–0282 T	Magnolia sp.	China	JX398982	JX399048	JX399017
N. sichuanensis	CFCC 54338 T	Castanea mollissima	China	MW166231	MW199750	MW218524
N. siciliana	AC46	Persea americana	Italy	ON117813	ON107273	ON209162
N. sonneratiae	MFLUCC 17-1745 T	Sonneronata alba	Thailand	MK764280	MK764324	MK764346
N. steyaertii	IMI 192475 T	Eucalytpus viminalis	Australia	KF582796	KF582792	KF582794
N. surinamensis	CBS 450.74 T	Soil under Elaeis guineensis	Suriname	KM199351	KM199518	KM199465
N. thailandica	MFLUCC 17-1730 T	Rhizophora mucronata	Thailand	MK764281	MK764325	MK764347
N. umbrinospora	MFLUCC 12–0285 T	unidentified plant	China	JX398984	JX399050	JX399019
N. vaccinii	CAA1059 T	Vaccinium corymbosum	Portugal	MW969747	MW959099	MW934610
N. vheenae	BRIP 72293a T	Macadamia integrifolia	Australia	MZ303792	MZ344177	MZ312685
N. vitis	MFLUCC 15-1265 T	Vitis vinifera	China	KU140694	KU140676	KU140685
N. zakeelii	BRIP 72282a T	Macadamia integrifolia	Australia	MZ303789	MZ344174	MZ312682
N. zimbabwana	CBS 111495 T	Leucospermum cuneiforme	Zimbabwe	N/A	KM199545	KM199456
Pestalotiopsis colombiensis	CBS 118553 T	Eucalyptus grandis × urophylla	Colombia	KM199307	KM199488	KM199421
Pestalotiopsis diversiseta	MFLUCC 12–0287 T	Dead plant material	China	NR_120187	JX399073	JX399040
Note: ¹ BRIP: Queensland Plant Pathology Herbarium, Australia; CAA: Personal culture collection of Artur Alves, Department of Biology, University of Aveiro; CBS: Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CFCC: China Forestry Culture Collection Center, Research Institute of Forest Ecology, Environment and Protection, Beijing, China; CGMCC: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; COAD: Culture collection of Coleção Octávio Almeida Drummond of the Universidade Federal de Viçosa, Viçosa, Brazil; CRM: Universidad Autónoma Chapingo, Centro Regional Morelia, Morelia, Michoacán, México; CSUFTCC: Central South University of Forestry and Technology culture collection, Hunan, China; FMBCC: Fungal Molecular Biology Laboratory Culture Collection, University of Agriculture Faisalabad, Pakistan; GUCC: Department of Plant Pathology culture collection, Agriculture College, Guizhou University, China; HGUP: Plant Pathology Herbarium of Guizhou University, Guizhou, China; IMI: Culture collection of CABI Europe UK Centre, Egham, UK; KUMCC: Culture collection of Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China; MFLUCC: Mae Fah Luang University culture collection, Chiang Rai, Thailand; TAP: Culture collection of Tamagawa University, Tokyo, Japan; URM: The Father Camille Torrend Herbarium, Pernambuco, Brazil. Ex-type strains are labeled with T. ² ITS: internal transcribed spacer; TEF: translation elongation factor 1-α; TUB: β-tubulin. N/A: Not available.

Phylogenetic analysis

The three genes present for all species included were aligned separately using the Mafft webserver (https://mafft.cbrc.jp/alignment/server/; Katoh et al. 2019). The three alignments were manually trimmed to remove leading and trailing gaps with MEGA v. 7 and subsequently merged to a final alignment with a length of 1710 positions. This was optimized using ModelFinder (Kalyaanamoorthy et al. 2017) and setting the branch length parameter to unlinked. Phylogenetic reconstructions were done using the Bayesian Inference (BI) algorithm as well as maximum likelihood (ML). The Bayesian analyses (MrBayes v3.2.7a, Ronquist et al. 2012) of four simultaneous Markov Chain Monte Carlo (MCMC) chains were run from random trees for 10 000 000 generations and sampled every 1 000 generations. The temperature value was lowered to 0.15, burn-in was set to 0.25, and the run was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01. Maximum likelihood analyse was conducted using IQ-TREE (version 2.2, Nguyen et al. 2015) applying a partitioned analysis with the best scheme from ModelFinder. All other parameters were set to default and bootstrapping (Felsenstein 1985) was performed with 1000 non-parametric replicates.

Pathogenicity tests

Mature, healthy leaves of zedoary turmeric underwent a preliminary sterilization step using 75% ethanol and rinsed three times with sterilized distilled water. The pathogenicity assessment was executed utilizing a specific isolate (CP1), whereby circular mycelial discs measuring 6 mm in diameter were extracted from a 7-day-old culture grown on PDA. These discs were strategically positioned on four distinct sites across each healthy leaf, both intact and wounded. For the purpose of wounding, 4 to 6 micropores were generated by piercing the leaf with an aseptic inoculation needle. Subsequently, the petiole was enveloped with absorbent cotton and introduced into a ceramic container, placed atop a layer of gauze. A minimal quantity of water was added into the bottom of the plate, which was subsequently sealed using cling film to keep it moist environment. A total of five zedoary turmeric leaves underwent the inoculation procedure, which was iterated three times. Concurrently, a sterile PDA block served as the negative control. Following the successful inoculation of zedoary turmeric leaves, the pathogenic fungus introduced during the process was subsequently re-isolated from the generated lesions. A comprehensive comparison ensued between the characteristics of the re-isolated fungus and the original CP1 strain.

Field symptom survey

In 2017, the novel occurrence of zedoary turmeric (Curcuma phaeocaulis) leaf blight emerged within the planting area of Nanxiong village, situated in Guilin city, Guangxi Province. The pathogenic impact was confined exclusively to the foliage, evoking discernible symptoms exclusively at the tips and margins of the upper and central leaves. During the initial stages of the disease progression, minute yellow‒green lesions manifested on the leaf surfaces. Subsequently, over a span of nearly 30 days, these lesion expanded toward leaf's tip or periphery, assuming a central brown hue with a yellowish border. A period of two months saw the gradual spread of these lesions to encompass the entirety of the leaf. Color transitions were noted from deep yellow at the periphery, evolving sequentially through pale brown to dark brown at the center. Concurrently, minuscule black speckles emerged on the lesion's surface. Clear demarcation materialized between afflicted and unaffected leaf regions. The ailment's exacerbation ultimately led to the demise of the entire plant (Fig. 1).

Phylogenetic analysis

The combined ITS, TEF and TUB alignment comprises 70 strains (including 68 strains for species of Neopestalotiopsis, two strains for species of Pestalotiopsis as the outgroup taxon) and 1710 characters (480 nucleotides of ITS, 865 nucleotides of TEF, and 365 nucleotides of TUB). The phylogeny of the individual loci (ITS, TEF and TUB) did not yield support for conflicting topologies and, thus, all loci were concatenated for phylogenetic analysis. Suitable models were selected using models of nucleotide substitution for each gene, as determined using ModelFinder. The HKY + F + I + G4 model with a proportion of invariable sites for ITS and the HKY + F + G4 model with gamma-distributed rate model for TEF and the K2P + G4 model with inverse gamma rate were selected for TUB and included for each gene partition. The Bayesian analysis lasted 10 000 000 generations and the 100% consensus trees and posterior probabilities were calculated from the 7501 trees left after discarding 2500 trees (the first 25% of generations) for burn-in. Phylogenetic reconstructions in Bayesian Inference (BI) and maximum likelihood (ML) resulted in largely congruent topologies with no support for conflicting topologies. A Thus, only the tree from the BI analysis is shown in Fig. 2, with bootstrap support (BS) from BI and ML inference added on the branches in the respective order. Within this clade the Strain CP1 and N. asiatica (MFLUCC 12–0286 and P817) were grouped together with strong support in both analyses.

Neopestalotiopsis asiatica Maharachch., K.D. Hyde and Crous, in Maharachchikumbura, Hyde, Groenewald, Xu and Crous, Stud. Mycol. 79: 147 (2014) (Fig. 3).

Conidiophores indistinct. Conidia 18.6–33.8×2.6–7.6 µm (‾х = 24.95 × 5.24), fusiform, straight to slightly curved, 4-septate; basal cell conical, hyaline, thin and verruculose, 3.5–6.8 µm long (‾х = 4.89 µm); three median cells 12.4–19.8 µm long (‾х = 17.05), dark brown, verruculose, septa and periclinal walls darker than the rest of the cell, versicoloured; apical cell 2.4–7.1 µm long (‾х = 4.71 µm), hyaline, conical to cylindrical, comprising 2–4 appendages (mainly 3); apical appendages 11.9–28.8 µm long (‾х = 20.6 µm), tubular, arising from the apex of the apical cell; basal appendage, 2.5–13.6 µm long (‾х = 5.9µm), filiform.

Culture characteristics. Colonies on PDA reaching 9 cm diam. after 7 days at 25°C, with crenate edge, whitish, with aerial mycelium on surface; fruiting bodies black, gregarious; reverse of culture whitish to pale yellow.

Habitat/Distribution: On the leaves of zedoary turmeric, Nanxiong, Guilin city, Guangxi Province China.

Notes. Our strain CP1 has identical highly similar ITS (99.36%) and TEF (99.79%) and TUB (98.92%) sequences to the type strain of N. asiatica (MFLUCC 12–0286). Neopestalotiopsis asiatica has been recorded as a pathogen of various plants, e.g., Vitis vinifera L. and Photinia × fraseri. The descriptions and illustrations of the other reports of N. asiatica cited above similar well with our strain, as do the molecular data.

Pathogenicity test

The results of the pathogenicity test showed that the specie of Neopestalotiopsis identified in this study were pathogenic to zedoary turmeric and produced the same symptoms similar to those observed in the field. Notably, 24 h following wound inoculation with strain CP1, initial lesion development was characterized by water-soaked areas, distinctly demarcating the boundary between diseased and healthy tissue. In parallel, under analogous experimental conditions, leaves subjected to non-wound inoculation manifested light brown lesions 48 h post inoculation (Fig. 4). The symptomatology exhibited by all inoculated leaves was consistent with that observed in the original ailment. Subsequent isolation of the pathogen from these lesions aligned seamlessly with the initially inoculated pathogen. In stark contrast, negative control leaves remained devoid of any discernible symptoms.

In this study, the pathogenicity of the strain CP1 to zedoary turmeric was established through a rigorous pathogenicity test following Koch's postulates. The isolate was observed to form a distinctive clade within the Neopestalotiopsis asiatica within a phylogenetic tree constructed from concatenated ITS, TEF, and TUB sequence data. In conclusion, our investigation elucidated that the Neopestalotiopsis asiatica strain CP is the causal agent responsible for inducing leaf blight symptoms in zedoary turmeric.

Conidium morphology is an extensively used taxonomic character in fungi (Maharachchikumbura et al. 2011). However, phenotypic characteristics overlap which makes it difficult to segregate morphologically equivocal taxa. Sibling taxa can be better resolved using sequence data. The ITS is the universal barcode for fung (Schoch et al. 2012). However, ITS could not fulfill the role as candidate gene for species discrimination, as the data did not have high variation between species. Cryptic taxa can be better resolved using slow evolving protein coding genes like TEF and TUB (Baldauf et al. 2000, Liu et al. 2010). Combined sequence analysis of ITS, TEF and TUB genes successfully resolved most of the Neopestalotiopsis species used in this study with high bootstrap support (Maharachchikumbura et al. 2014).

Leaf diseases in zedoary turmeric have been attributed to a spectrum of pathogens. For instance, Phomopsis longicolla results in the formation of yellow water-soaked spots, originating at the leaf tip or margin, which progressively expand to the primary vein and ultimately adopting a "V" shaped spot (Jiang et al. 2016). Colletotrichum curcumae, the instigator of anthracnose disease in Curcuma wenyujin, induces irregular yellowish-brown lesions on leaves devoid of black speckling (Li et al. 2016), while Curvularia clavata, responsible for Curcuma wenyujin leaf blight, provokes lesions at the tip and main vein, resulting in overall plant decline (Chen et al. 2013). Intriguingly, our findings indicate a distinct absence of prior reports detailing zedoary turmeric leaf blight symptoms attributable to Neopestalotiopsis, distinct from those instigated by other pathogens. As such, we propose labeling this ailment as zedoary turmeric leaf blight. It's noteworthy that Neopestalotiopsis demonstrated its capacity to impact a range of plant species including macadamia nuts, strawberries and rubber trees.

Presently, chemical control strategies predominantly encompass the management of Neopestalotiopsis species. For instance, the fungicide SYP-14288 (Shengyang Research Institute of Chemical Industry, Shengyang), has exhibited effectiveness in curbing strawberry leaf spot caused by N. clavispora (Wen et al. 2019). Difenoconazole and thiram have been identified as inhibitors of hyphal growth of N. clavispora, with thiram additionally impeding conidial germination and mitigating root rot and leaf spot in blueberry (Xue et al. 2018). Our study introduces the leaf blight of zedoary turmeric elicited by Neopestalotiopsis asiatica strain CP1, marking a pioneering observation. Nevertheless, the identification of an efficacious fungicidal treatment for disease control remains elusive, warranting further exploration.

ACKNOWLEDGEMENTS

We gratefully acknowledge Prof. Yan Zhou (Citrus Research Institute, China) for giving some information to this research and revise this paper.

FUNDING

This work was supported by China Agriculture Research System (Grant No. CARS-21)

Availability of data and materials

All data generated during the study are interpreted in the manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Neopestalotiopsis asiatica strain CP1 Causing Leaf Blight on Zedoary Turmeric in Guangxi, China

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Abstract

Background

Results

Conclusions

Figures

INTRODUCTION

MATERIALS AND METHODS

Field survey and fungal isolation

Morphological characteristics

DNA extraction, polymerase chain reaction (PCR) and sequencing

Phylogenetic analysis

Pathogenicity tests

RESULTS

Field symptom survey

Phylogenetic analysis

Pathogenicity test

DISCUSSION

Declarations

References

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