Fungal Pathogens on Bast Fiber Crops

Fungal Pathogens on Bast Fiber Crops: History

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Mycology | Agronomy

Contributor: Jianping Xu

Bast fiber crops are an important group of economic crops for the purpose of harvesting fibers from stems. These fibers are sclerenchyma fibers associated with the phloem of plants. They arise either with primary tissues from the apical meristem, or with secondary tissues produced by the lateral meristem. Fungal diseases have become an important factor limiting their yield and quality, causing devastating consequences for the production of bast fiber crops in many parts of the world.

bast fiber crops
molecular identification
fungal disease
DNA barcode
PCR assay

1. Introduction

Plant infectious diseases are among the most important constraints on the quality and yield of crops. It is estimated that plant diseases cause losses of 10%–15% of the world’s major crops, with direct economic losses of up to hundreds of billions of dollars each year. About 70%–80% of crop diseases are caused by fungal pathogens and the damage can be very serious, significantly reducing the yield and quality of many staple food crops and economic crops like fruits, vegetables, and fiber crops ^[1]. In addition, several fungal pathogens can secrete a variety of toxins and metabolites harmful to humans and animals, posing a great threat to the safety of agricultural products ^[2]. At present, most control measures against plant fungal pathogens rely on the applications of broad-spectrum fungicides. However, such fungicides not only increase production costs, but also can bring problems such as environmental pollution, fungicide resistance, and persistent residues on foods and other consumer goods with further implications for human health. In order to minimize the damage to crops caused by fungal diseases, as well as to maximize productivity and ensure agricultural sustainability, early detection and quantification of fungal pathogens is essential for disease prevention and control. However, conventional protocols based on morphological and physiological methods are time-consuming, require significant experience, and may not be sensitive and specific for individual pathogens ^[3]. Moreover, many fungal pathogens can remain latent in “sub-infection” stages with no obvious symptoms and/or in low numbers, making them difficult to detect, and causing confusion with regard to their roles in diseases. These issues can contribute to delayed or wrong control measures.

During the last three decades, to overcome these problems and minimize crop losses caused by fungal diseases, a diversity of DNA molecule-based tools has been developed for the detection and identification of fungal pathogens. These techniques include conventional polymerase chain reaction (PCR) ^[4], quantitative PCR (qPCR) ^[5]^[6], immunocapture-PCR (IC-PCR) ^[7]^[8], droplet digital PCR (dd-PCR) ^[9], loop-mediated isothermal amplification (LAMP) ^[10], multiplex tandem PCR ^[11], fluorescence in situ hybridization (FISH) ^[12], and DNA microarrays ^[3]. These methods are typically faster and more accurate than those based on colony morphology, microscopic features, and/or physiological/biochemical characters of pure fungal cultures. Indeed, methods targeting DNA sequences have been applied to detect pathogens during crops’ growth, harvest and post-harvest processing stages ^[13]. Moreover, they have also enabled a deeper understanding of microbial populations and communities associated with crops, especially the microorganisms that are difficult or impossible to cultivate in the lab. Together, technological advances and developments in DNA molecule-based methods have allowed fast and accurate detection and quantification of several fungal pathogens simultaneously in many important crops ^[14]^[15]. Information resulting from such work has been used to improve disease control and prevention with more rational decisions about the choice of fungicides to use, the appropriate cultivar(s) to plant, and necessary sanitary measures to apply during various stages of the crop production and processing cycle ^[16]^[17]^[18]^[19].

2. Bast Fiber Crops

Bast fiber crops are an important group of economic crops for the purpose of harvesting fibers from stems ^[20]. These fibers are sclerenchyma fibers associated with the phloem of plants. They arise either with primary tissues from the apical meristem, or with secondary tissues produced by the lateral meristem. Bast fiber is one of four major types of natural plant fibers, with the other three being leaf fiber (e.g., banana and pineapple fibers), fruit and seed fiber (e.g., cotton and coconut fiber), and stalk fiber (e.g., straw fiber from rice, wheat, and bamboo). Bast fiber crops comprise six main species (flax, hemp, ramie, kenaf, jute, and sunn hemp) that are broadly cultivated (Table 1) as well as a few others (kudzu, linden, milkweed, nettle, okra, and paper mulberry) with more limited fiber production ^[21]. Table 1 summarizes the main bast fiber crops, including their geographic distributions, habitats, commercial use, and main fungal diseases.

Table 1. Major types of bast fiber crops and their distributions around the world ^[20]^[21]^[22].

Crop	Main Distribution	Main Characters of Growth Habitat	Main Applications	Main Fungal Diseases
Flax (Linum usitatissimum Linnaeus)	France, Russia, Netherlands, Belarus, Belgium, Canada, Kazakhstan, China, India	Well-drained loam and cool, moist, temperate climates	Linen, flax yarn, flax seed, linseed oil	flax wilt, flax blight, flax anthracnose
Hemp (Cannabis sativa Linnaeus)	China, Canada, USA, Europe, East Asia, Nepal	Grows at 16–27 °C, sufficient rain at the first six weeks of growth, short day length.	Textiles, hempseed oil, prescription drugs	hemp powdery mildew, hemp leaf spot disease, hemp blight, hemp root and crown rot wilt, hemp charcoal rot
Jute (Corchorus capsularis Linnaeus)	India, Bangladesh, Burma, China	Tropical lowland areas, humidity of 60% to 90%, rain-fed crop	Textiles, medicine	jute anthracnose, jute brown wilt, jute leaf spot
Kenaf (Hibiscus cannabinus Linnaeus)	India, Bangladesh, China, Malaysia, Thailand	Sandy loam and warm, humid subtropical, or tropical climates, few heavy rains or strong winds, at least 12 h light each day	Textiles	kenaf anthracnose, kenaf lack rot, kenaf sooty mold
Ramie (Boehmeria nivea Linnaeus) Gaudich	China, Brazil, Philippines, India, Vietnam, Laos, Cambodia	Sandy soil and warm, wet climates, rainfall averaging at least 75 to 130 mm per month	Textiles, soil and water conservation, medicine	ramie anthracnose, ramie powdery mildew, ramie black leaf spot, ramie blight
Sunn Hemp (Crotalaria juncea Linnaeus)	India, USA, China	Wide variety of soil condition, altitude from 100 to 1000 m, temperatures above 28 °C, photoperiod-sensitive	Cover crop or green manure, forage producer	sunn hemp fusarium wilt, sunn hemp root rot, sunn hemp powdery mildew

3. Fungal Pathogens of Bast Fiber Crops

As shown in Table 1, most bast fiber crops can grow in a diversity of geographic regions and ecological niches. However, some of them have relatively limited geographic and/or ecological distributions and can’t grow well in certain environments. As a result, the types of land used to cultivate certain bast fiber crops may be limited and the same fields may be used to grow the same crop over many years. Even for bast fiber crops with broad ecological adaptability, the limited agricultural land in certain regions and the drive to seek high commercial benefits often mean that only certain types of fields are used for growing each specific crop. In these fields, fungal infectious diseases often increase over time, leading to large yield loss, or even total destruction of the harvest. Fungal pathogens occurring on bast fiber crops are taxonomically very broad (Table 2).

Table 2. List of fungal pathogens on bast fiber crops identified using molecular method.

Pathogen		Disease	Method	Marker	Host Plant	Geographic Region(s)	Reference
Alternaria
A. alternata		Hemp leaf spot	Conventional PCR	ITS	Cannabis sativa	Shanxi, China	^[23]
A. alternata		Ramie black leaf spot	Conventional PCR	ITS, GAPDH	Boehmeria nivea	Hunan, Hubei, China	^[24]
Cercospora
Cercospora cf. flagellaris		Hemp leaf spot disease	Not mentioned	ITS, EF-1α, CAL, H3, actin	Cannabis sativa	Kentucky, USA	^[25]
Colletotrichum
C. corchorum capsularis		Jute anthracnose	Conventional PCR	ACT, TUB2, CAL, GAPDH, GS, and ITS	Corchorus capsularis L.	Zhejiang, Fujian, Guangxi, and Henan, China	^[26]
C. fructicola		Jute anthracnose	Conventional PCR	ACT, TUB2, CAL, GAPDH, GS, and ITS	Corchorus capsularis L.	Zhejiang, Fujian, Guangxi, and Henan, China	^[27]
C. fructicola		Jute anthracnose	Conventional PCR	ACT, TUB2, CAL, GAPDH, GS, and ITS	Corchorus capsularis L.	Zhejiang, Fujian, Guangxi, and Henan, China	^[26]
C. gloeosporioides		Ramie anthracnose	Conventional PCR	ITS	Boehmeria nivea	HuBei, HuNan, JiangXi, and SiChuan, China	^[28]
C. higginsianum		Ramie anthracnose	Conventional PCR	ITS	Boehmeria nivea	HuBei, China	^[29]
C. phormii		New Zealand flax anthracnose	Conventional PCR	ITS	Phormium tenax	California, USA	^[30]
C. phormii		New Zealand flax anthracnose	Conventional PCR	ITS	Phormium tenax	Perth, Australia	^[31]
C. siamense		Jute anthracnose	Conventional PCR	ACT, TUB2, CAL, GAPDH, GS, and ITS	Corchorus capsularis L.	Zhejiang, Fujian, Guangxi, and Henan, China	^[27]
Colletotrichum sp.		Kenaf anthracnose	Conventional PCR	ITS	Corchorus olitorius	South Korea	^[32]
Curvularia
C. cymbopogonis		Hemp leaf spot	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Exserohilum
E. rostratum		Hemp floral blight	Not mentioned	ITS, RPB2	Cannabis sativa	North Carolina, USA	^[34]
Fusarium
F. oxysporum		Hemp roots and crown rot	Conventional PCR	ITS, EF-1α	Cannabis sativa	Canada	^[35]
F. oxysporum		Jute brown wilt	Conventional PCR	ITS	Corchorus olitorius	Dhaka, Manikgonj, Kishorgonj, Rangpur, and Monirampur, Bangladesh	^[36]
F. oxysporum		Hemp wilt	Conventional PCR	ITS, EF-1α	Cannabis sativa	California, USA	^[37]
F. solani		Hemp crown root	Conventional PCR	ITS, EF-1α	Cannabis sativa	Canada	^[35]
F. solani		Hemp wilt	Conventional PCR	ITS, EF-1α	Cannabis sativa	California, USA	^[37]
F. solani		Sunn hemp root rot and wilt	Conventional PCR	ITS, EF-1α	Crotalaria juncea	Ceará, Brazil	^[38]
F. brachygibbosum		Hemp wilt	Conventional PCR	ITS, EF-1α	Cannabis sativa	California, USA	^[37]
F. udum f. sp. crotalariae		Sunn hemp fusarium wilt	Conventional PCR	EF-1α, β-tubulin	Crotalaria juncea	Tainan, China	^[39]
Glomus
G. mosseae		Hemp root rot	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Golovinomyces
G. spadiceus		Hemp powdery mildew	Not mentioned	ITS, 28S	Cannabis sativa	Kentucky, USA	^[40]
G. cichoracearum sensu lato		Hemp powdery mildew	Conventional PCR	ITS	Cannabis sativa	Atlantic Canada and British Columbia.	^[41]
G. cichoracearum	Sunn hemp powdery mildew		Not mentioned	ITS	Crotalaria juncea	Florida, USA	^[42]
Lasiodiplodia
L. theobromae		Kenaf black rot	Conventional PCR	ITS	Corchorus olitorius	Kangar Perlis, Malaysia	^[43]
Leptoxyphium
L. kurandae		Kenaf sooty mould	Conventional PCR	ITS	Corchorus olitorius	Iksan, Korea	^[44]
Macrophomina
Macrophomina phaseolina		Hemp charcoal rot	Conventional PCR	EF-1α, CAL	Cannabis sativa	Southern Spain	^[45]
Micropeltopsis
Micropeltopsis cannabis		Unknown	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Orbilia
Orbilia luteola		Unknown	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Pestalotiopsis
Pestalotiopsissp.		Hemp spot blight	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Podosphaera
P. xanthii		Ramie powdery mildew	Conventional PCR	ITS	Boehmeria nivea	Naju, Korea	^[46]
Pythium
P. dissotocum		Browning and a reduction in root mass, stunting	Conventional PCR	ITS, EF-1α	Cannabis sativa	Canada	^[35]
P. myriotylum		Browning and a reduction in root mass, stunting	Conventional PCR	ITS, EF-1α	Cannabis sativa	Canada	^[35]
P. myriotylum		Hemp root rot and Wilt	Conventional PCR	ITS, COI, COII	Cannabis sativa	Connecticut, USA	^[47]
P. aphanidermatum		Hemp root rot and crown wilt	Conventional PCR	ITS	Cannabis sativa	California, USA	^[37]
P. aphanidermatum		Hemp crown and root Rot	Conventional PCR	ITS	Cannabis sativa	Indiana, USA	^[48]
P. ultimum		Hemp crown and root Rot	Conventional PCR	ITS	Cannabis sativa	Indiana, USA	^[49]
Rhizoctonia
Binucleate R. spp.		Hemp wilt	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Sclerotinia
Sclerotinia minor		Hemp crown rot	Conventional PCR	ITS	Cannabis sativa	San Benito County, Canada	^[50]
Sphaerotheca
S. macularis		Hemp powdery mildew	Conventional PCR	25S	Cannabis sativa	USA	^[33]
Verticillium
V. dahliae		flax wilt	Conventional PCR	ITS	Linum usitatissimum	La Haye Aubrée, France	^[51]
V. dahliae		flax wilt	qPCR	ITS	Linum usitatissimum	Normandy, France	^[52]
V. dahliae		flax wilt	qPCR	ß-tubulin	Linum usitatissimum	Germany	^[53]
V. tricorpus		flax wilt	qPCR	ITS	Linum usitatissimum	Germany	^[53]
V. longisporum		flax wilt	qPCR	ß-tubuIin	Linum usitatissimum	Germany	^[53]

qPCR: quantitative PCR, ITS: internal transcribed spacer, GAPDH: glyceraldehydes-3-phosphate dehydrogenase, GS: glutamate synthetase, EF-1α: translation elongation factor 1-α, CAL: calmodulin, H3: histone subunit 3, ACT: actin, TUB2: ß-tubulin, RPB2: RNA polymerase subunit B2, COI: cytochrome oxidase subunit I, COII: cytochrome oxidase subunit II.

4. Development of Molecular Identification of Bast Fiber Fungal Pathogens

At present, most diagnosis of bast fiber diseases rely on disease symptoms and, when available, cultural characteristics of isolated fungal pathogens on artificial media. However, it is often difficult to identify the underlying pathogen based on those characters alone. For example, the disease symptoms of Verticillium wilt in hemp is very similar to Fusarium wilt and the pathogen species in both genera can invade a wide range of economical crops ^[51]^[52]^[53]. In addition, it is difficult to distinguish the species within most fungal genera based on morphological features alone. However, most of them are relatively easy to identify using molecular markers, as described below (Table 2; Table 3).

Table 3. Genes and PCR primers used for their amplification in fungal pathogens infecting bast fiber crops.

Target DNA	Primer Name and Sequence (5′-3′)		Size of PCR Product (bp)	Reference
18S	NS3	GCAAGTCTGGTGCCAGCAGCC	Not mentioned	^[54]
18S	NS4	CTTCCGTCAATTCCTTTAAG	Not mentioned	^[54]
28S	LR0R	GCAAGTCTGGTGCCAGCAGCC	Not mentioned	^[54]
28S	LR3	GCAAGTCTGGTGCCAGCAGCC	Not mentioned	^[54]
25S	LROR	ACCCGCTGAACTTAAGC	1431	^[33]
25S	LR7	TACTACCACCAAGATCT	1431	^[33]
ACT	ACT-512F	ATGTGCAAGGCCGGTTTCGC	300	^[25]
ACT	ACT-783R	TACGAGTCCTTCTGGCCCAT	300	^[25]
ß-tubulin	Vd-btub-1F	GCGACCTTAACCACCTCGTT	Not mentioned	^[52]
	Vd-btub-1R	CGCGGCTGGTCAGAGGA	Not mentioned	^[52]
	VertBt-F	AACAACAGTCCGATGGATAATTC	Not mentioned	^[52]
	VertBt-R	GTACCGGGCTCGAGATCG	Not mentioned	^[52]
	VITubF2	GCAAAACCCTACCGGGTTATG	143	^[53]
	VITubRl	AGATATCCATCGGACTGTTCGTA	143	^[53]
	VdTubF2	GGCCAGTGCGTAAGTTATTCT	82	^[53]
	VdTubR4	ATCTGGTTACCCTGTTCATCC	82	^[53]
	Bt2a	GGTAACCAAATCGGTGCTGCTTTC	Not mentioned	^[27]
	Bt2b	ACCCTCAGTGTAGTGACCCTTGGC	Not mentioned	^[27]
CAL	CL1	GARTWCAAGGAGGCCTTCTC	Not mentioned	^[27]
	CL2	TTTTTGCATCATGAGTTGGAC	Not mentioned	^[27]
	CAL-228F	GAGTTCAAGGAGGCCTTCTCCC	Not mentioned	^[45]
	CAL-737R	CATCTTTCTGGCCATCATGG	Not mentioned	^[45]
EF-1α	EF-1	ATGGGTAAGGAGGACAAGAC	700	^[37]
	EF-2	GGAGGTACCAGTGATCATGTT	700	^[37]
	EF1-728F	CATCGAGAAGTTCGAGAAGG	Not mentioned	^[45]
	EF2	GGAGGTACCAGTGATCATGTT	Not mentioned	^[45]
	EF1-728F	CATCGAGAAGTTCGAGAAGG	350	^[25]
	EF1-983R	TACTTGAAGGAACCCTTACC	350	^[25]
Endochitinase	Vd-endoch-1F	CTCGGAGGTGCCATGTACTG	Not mentioned	^[52]
Endochitinase	Vd-endoch-1R	ACTGCCTGGCCCAGGTTC	Not mentioned	^[52]
GAPDH	Vd-G3PD-2F	CACGGCGTCTTCAAGGGT	Not mentioned	^[52]
	Vd-G3PD-1R	CAGTGGACTCGACGACGTAC	Not mentioned	^[52]
	GDF1	GCCGTCAACGACCCCTTCATTGA	Not mentioned	^[27]
	GDR1	GGGTGGAGTCGTACTTGAGCATGT	Not mentioned	^[27]
	gpd-1	CAACGGCTTCGGTCGCATTG	Not mentioned	^[24]
	gpd-2	GCCAAGCAGTTGGTTGTGC	Not mentioned	^[24]
GS	GSF1	ATGGCCGAGTACATCTGG	Not mentioned	^[27]
GS	GSR1	GAACCGTCGAAGTTCCAC	Not mentioned	^[27]
ITS	ITS1	TCCGTAGGTGAACCTGCGG	334-738	^[30]^[28]^[48]^[49]^[51]^[52]
	ITS4	TCCTCCGCTTATTGATATGC	334-738	^[30]^[28]^[48]^[49]^[51]^[52]
	Vd-ITS1-45-F	CCGGTCCATCAGTCTCTCTG	334	^[51]
	Vd-ITS2-379-R	ACTCCGATGCGAGCTGTAAC	334	^[51]
	ITS1-F	CTTGGTCATTTAGAGGAAGTAA	700	^[37]
	ITS4	TCCTCCGCTTATTGATATGC	700	^[37]
	VtF4	CCGGTGTTGGGGATCTACT	123	^[53]
	VtR2	GTAGGGGGTTTAGAGGCTG	123	^[53]
	ITS 4	TCCTCCGCTTATTGATATGC	Not mentioned	^[27]
	ITS 5	GGAAGTAAAAGTCGTAACAAGG	Not mentioned	^[27]
RPB2	bRPB2-6F	TGGGGYATGGTNTGYCCYGC	Not mentioned	^[34]
RPB2	bRPB2-7R	GAYTGRTTRTGRTCRGGGAAVGG	Not mentioned	^[34]

As early as 1997, a PCR-based method was used to help identify fungal pathogens of bast fiber crops. Specifically, McPartland et al. ^[33] amplified part of the 28S ribosomal RNA (rRNA) gene followed by EcoR I/Hind III digestion and electrophoresis to differentiate hemp fungal pathogens, and named two new species: Micropeltopsis cannabis sp. nov. and Orbilia luteola (Roum.) comb. nov. However, there were relatively few reports of fungal pathogens on bast fiber crops between 1998 and 2009, likely due to limited production of bast fiber crops and an emphasis on chemical fiber and other natural fibers. During this period, the acreage and production of bast fiber crops were low and there was limited research on these crops. Since 2009, with increasing production and research on bast fiber crops, there have been increasing reports on infectious diseases, including fungal diseases, on these crops ^[55]. This is especially true over the last five years when a large number of fungal pathogens were reported from bast fiber crops and many of these were identified based on molecular markers (Figure 1).

Figure 1. Development of molecular-based assays for the detection of fungal pathogens in bast fiber crops from 1997 until the present. For genus and species names, please see text and Table 2. Details of primers are shown in Table 3.

According to the National Center for Biotechnology Information (NCBI) PubMed, the most common literature on the molecular identification of fungal pathogens on bast fiber crops has been on hemp (including both industrial hemp and medicinal marijuana), accounting for ~45% of all published articles. This was then followed by flax and kenaf (at ~14% each), ramie (11%), and the rest being jute and sunn hemp. However, most of these reports were case reports.

5. Target DNA Selection and Molecular Assays of Fungal Pathogens on Bast Fiber Crops

Over the last three decades, several types of DNA-based methods have been developed and widely used to detect plant fungal pathogens. The invention of PCR technology using a thermostable polymerase by Kary Mullis gave birth to PCR in the early 1980s ^[4]. The invention of PCR has led to a diversity of PCR-based methods for fungal pathogen detections based on variations in DNA sequences within and among species (Figure 1, Table 2). Among these methods, qPCR is probably the most common molecular technology and it can be used for quantitative measurement of RNA and DNA, targeting both single nucleotide polymorphisms (SNPs) and copy number variations. qPCR allows not only the detection of whether a specific pathogen(s) is present in the sample, but also the quantification of pathogen levels in host tissues ^[5]^[6]. To improve the efficiency of conventional PCR, other methods have been coupled with PCR for plant fungal pathogen detection. For example, PCR in combination with enzyme-linked immunosorbent assay (ELISA) has been successfully applied to detect fungi, viruses, and bacteria, with high specificity ^[56]. Similarly, the highly specific IC-PCR approach can increase the sensitivity by 250 folds compared to conventional PCR amplification ^[7]^[8]. For absolute quantification without the need for references and standard curves, dd-PCR is the method of choice—this method is based on the combined technology of water–oil emulsion droplet and PCR ^[9]. In field conditions without ready access to laboratory equipment, LAMP can provide fast identifications of samples. LAMP uses six primers that are highly specific to target sites in a specific gene ^[10]. It can be carried out at a constant temperature in a short reaction time (<30 min). It is sensitive and cost-effective, potentially making it an ideal method for field detection of plant pathogens ^[57].

As shown in Table 2, PCR-based methods have been used as the main approach for detecting fungal pathogens in bast fiber crops. This pattern is similar to the detections of fungal pathogens in other crops in general. A number of DNA fragments and genes have been explored as potential targets for PCR-based detections, including the ribosomal RNA gene cluster, conserved housekeeping genes, and genes involved in the production of secondary metabolites, including mycotoxins ^[58]^[59]^[60]. Table 3 summarizes the genes and their primers that have been used for the detection and diagnostics of fungal pathogens on bast fiber crops. The researchers would like to note that the molecular analyses reported so far for identifying fungal pathogens on bast fiber crops have been primarily using pure fungal strains, not those from diseased plant tissues. There is a large gap in applying these molecular methods in field conditions as a point-of-care test.

Among the DNA fragments that have been used for fungal pathogen detection, the most frequently used is the ribosomal RNA gene cluster. This gene cluster is composed of up to hundreds of repeating units with each unit containing the genes encoding the small (18S) ribosomal RNA subunit, the internal transcribed spacer (ITS) regions 1 and 2 that are separated by the 5.8S rRNA subunit, and the large (28S) ribosomal RNA subunit, with the intergenic spacer (IGS) region separating the adjacent units (Figure 2). The entire ITS fragment (which comprises ITS1, 5.8S rRNA, and ITS2) is typically 500–750 bp long and flanked by the 18S and 28S rRNA genes ^[61]^[62]^[63]. The ITS regions are present in all known fungi and have both highly conserved flanking regions located in the 5.8S, 18S, and 28S rRNA genes as well as the variable regions (located in the ITS1 and ITS2 regions). The conserved flanking regions allowed the development of highly conserved probes or primers to amplify most, if not all, fungi, while the variable regions allowed the development of species-specific markers ^[64]^[65]. Together, these features have contributed to ITS being the consensus fungal DNA barcode for the mycological community ^[64]^[65]. Furthermore, the ITS sequences obtained from the direct amplification and sequencing of environmental DNA samples have contributed to our increased understanding of fungal diversity from a variety of environments, including those from diseased plants and animals ^[65]^[66].

Figure 2. A schematic representation of the fungal ribosomal RNA gene cluster showing the locations of individual DNA fragments and the common primers used for PCR amplification.

This entry is adapted from the peer-reviewed paper 10.3390/pathogens9030223

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