Morphological and molecular identification of Cladosporium sphaerospermum isolates collected from tomato plant residues

Identificação morfológica e molecular de isolados de Cladosporium sphaerospermum coletados de resíduos de plantas de tomate

Abstract

This study was conducted at the Agriculture College University of Karbala, Iraq to isolate and morphologically and molecularly diagnose thirteen Cladosporium isolates collected from tomato plant residues present in desert regions of Najaf and Karbala provinces, Iraq. We diagnosed the obtained isolates by PCR amplification using the ITS1 and ITS4 universal primer pair followed by sequencing. PCR amplification and analysis of nucleotide sequences using the BLAST program showed that all isolated fungi belong to Cladosporium sphaerospermum. Analysis of the nucleotide sequences of the identified C. sphaerospermum isolates 2, 6, 9, and 10 showed a genetic similarity reached 99%, 98%, 99%, and 99%, respectively, with those previously registered at the National Center for Biotechnology Information (NCBl). By comparing the nucleotide sequences of the identified C. sphaerospermum isolates with the sequences belong to the same fungi and available at NCBI, it was revealed that the identified C. sphaerospermum isolates 2, 6, 9, and 10 have a genetic variation with those previously recorded at the National Center for Biotechnology Information (NCBl); therefore, the identified sequences of C. sphaerospermum isolates have been registered in GenBank database (NCBI) under the accession numbers MN896004, MN896107, MN896963, and MN896971, respectively.

Keywords:
Cladosporium sphaerospermum; PCR amplification; ITS1 and ITS4; fungi

Resumo

Este estudo foi conduzido na Agriculture College University of Karbala, Iraque, para isolar e diagnosticar morfológica e molecularmente treze isolados de Cladosporium coletados de resíduos de plantas de tomate presentes nas regiões desérticas das províncias de Najaf e Karbala, no Iraque. Diagnosticamos os isolados obtidos por amplificação por PCR usando o par de primers universais ITS1 e ITS4 seguido de sequenciamento. A amplificação por PCR e a análise de sequências de nucleotídeos usando o programa BLAST mostraram que todos os fungos isolados pertencem a Cladosporium sphaerospermum. A análise das sequências de nucleotídeos dos isolados 2, 6, 9 e 10 de C. sphaerospermum identificados mostrou similaridade genética de 99%, 98%, 99% e 99%, respectivamente, com aqueles previamente registrados no National Center for Biotechnology Informações (NCBl). Ao comparar as sequências de nucleotídeos dos isolados de C. sphaerospermum identificados com as sequências pertencentes aos mesmos fungos e disponíveis no NCBI, foi revelado que os isolados 2, 6, 9 e 10 de C. sphaerospermum identificados têm variação genética com aqueles anteriormente registrados no National Center for Biotechnology Information (NCBl). Portanto, as sequências identificadas de isolados de C. sphaerospermum foram registradas no banco de dados GenBank (NCBI) sob os números de acesso MN896004, MN896107, MN896963 e MN896971, respectivamente.

Palavras-chave:
Cladosporium sphaerospermum; amplificação por PCR; ITS1 e ITS4; fungos

1. Introduction

Fungi exist in all environmental systems and have different effects between them as well as with the other organisms (Engelthaler & Litvintseva, 2020). Cladosporium sphaerospermum was first described by Albert Julius Otto Penzig in 1886 after isolating from the rotting leaves and branches of citrus (Citrus aurantium) (Bensch et al., 2010). C. sphaerospermum is a cosmopolitan fungus and present in many internal and external environments because of its airborne and rapid spread nature (Zalar et al., 2007; Bensch et al., 2010). C. sphaerospermum was isolated from very salty environments in Mediterranean and tropical as well as from temperate climates (Zalar et al., 2007). Within the buildings, this fungus can extensively grow on the interior surfaces of windows, bathrooms, and kitchens (Hocking et al., 1994; Adan et al., 2011).

C. sphaerospermum can be isolated from decaying leaves, stems of woody and herbaceous plants, vegetables, and fruits (Dugan et al., 2018). In India, Avasthi et al. (2016) reported that C. sphaerospermum is the causal agent of leaf spot disease on Aloe vera plants. Plant tissues can be attacked by C. sphaerospermum causing dead or senescent as well as it can cause spoilage of various post-harvested fruits and vegetables, e.g. citrus, strawberry, apple, and tomato fruits leading to a decrease in nutritional and marketing values of these fruits (Qiu‐Xia et al., 2008).

There are very few reports implicating this species as a disease agent in humans (Hocking et al., 1994). C. sphaerospermum is a very rare cause of human sickness, but it can cause some different types of infections, including, sinus, skin, eye, and brain infections (Zalar et al., 2007; Maduri et al., 2015). It was reported that a female patient showed swelling on the dorsum of her hand and after testing with Lactophenol cotton blue, and Grocott's methenamine silver stain revealed the presence of hyphae compatible with the fungus C. sphaerospermum (Maduri et al., 2015). Another case reported where C. sphaerospermum caused cerebral phaeohyphomycosis but the patient was successfully treated and the symptoms were reduced (Lai et al., 2013). It was also mentioned that C. sphaerospermum can produce different allergenic compounds but has not been known to be able produce significant mycotoxins (Ng et al., 2012).

Morphological identification of fungi is still difficult and insufficient for identifying because some of the characteristics of the species are very similar to the characteristics in Cladosporium species (Dugan et al., 2008). Therefore, for accurate identification, various molecular methods have been successfully applied to diagnose many fungi such as Fusarium spp., Rhizoctonia solani, Aspergillus spp. (Salem et al., 2019; Abedalred et al., 2019). Among the molecular techniques used, the polymerase chain reaction (PCR) has been successfully applied to diagnose Cladosporium species using the internal transcribed spacer (ITS) that is the universal barcode of fungi and can be used for identification of Cladosporium spp. (Hensel et al., 2020; Han et al., 2020). ITS sequences are commonly used to construct the phylogenetic trees and identify fungi (Salem et al., 2019; Ao et al., 2020). Morphological identification and sequencing of ITS can be used to identify Cladosporium isolates to the species level. Therefore, the objective of this research was to diagnose some Cladosporium species isolated from tomato plant residues (roots and stems) using morphological and molecular characteristics.

2. Materials and Methods

2.1. Sampling and fungal isolation

Samples were collected from tomato residues (roots and stems) found in desert region farms in Najaf and Karbala provinces, Iraq and then brought to the laboratory of Plant Virology at Agriculture College in the Plant Protection Department, Karbala University for fungal isolation. Collected samples were washed by water, cut into small parts, sterilized with sodium hypochlorite solution (NaOCl 1%) for 2 min, and rinsed with distilled water. These pieces were dried using sterilized filter papers and transferred to the Petri dishes containing Potato Dextrose Agar medium (200 g potato, 20 g dextrose, 15 g agar, and 1 liter of water) supplemented with chloramphenicol antibiotic (200 mg/L). Inoculated Petri dishes were kept at 25 ± 2 °C for 3 days. Appeared fungal colonies were purified on PDA medium and initially diagnosed using the morphological characteristics. These isolates were also molecularly diagnosed by the PCR technology and determining the sequences generated from the PCR-amplified products according to the method given later.

Genomic DNA extraction, PCR amplification and sequencing of ITS region

From each isolate, about 100 mg of a 7-day-old colony were transferred using a sterile scalpel into an Eppendorf tube to extract DNA using a fungal/ bacteria DNA extraction kit (Zymo Research, Cat. No. D6005, USA) following the manufacturer’s instructions. The purity (A_260/280) and concentration (A₂₆₀) of DNA extracted from each fungal isolate were determined using a UV spectrophotometer (Thermo Scientific, Germany). Thereafter, DNA was stored at -20°C until use.

The ITS (internal transcribed spacer) region of C. sphaerospermum isolates was amplified using the primer pair ITS1 (TCCGTTGGTGAACCAGCGG) and ITS4 (TCCTCCGCTTATGATA TGC) (White et al., 1990) and the PCR mixture shown in Table 1. PCR amplification carried out using the following conditions: initial denaturation for 1 min at 94 °C followed by 35 cycles each consists of final denaturation at 94 °C for 30 s, annealing temperature for 30s at 55 °C, initial extension for 1 min at 72 °C, and final extension for 5 min at 72 °C (White et al., 1990). PCR products were run on a 1% agarose gel for 150 min at 80V and 400 mA, ethidium bromide stained and visualized under UV illumination. Gel pictures were taken using gel documentation system, Vilber Lourmat, Taiwan.

Table 1
Components used for PCR amplification.

Component	Volume
1× PCR buffer	2 μL
template DNA (30 ng/μL)	3 μL
dNTPs (2 mM)	2 μL
Taq polymerase	1 unit
Forward primer (10 pmol)	1 μL
Reverse primer (10 pmol)	1 μL
Nuclease-free sterile distilled water	10 μL

For nucleotide sequencing, the PCR products were gel-purified by the FavorPrep PCR Purification Kit (Taiwan, Favorgen, Cat. No. FAGCK 001) and sent with the ITS1 and ITS4 primer pair to the Macrogen DNA sequencing service in South Korea. PCR products were sequenced in both directions using the ITS1 and ITS4 primer pair. The nucleotide sequences were then aligned to compare with the sequences belonged to the C. sphaerospermum isolates deposited in NCBI using BLAST (Basic Local Alignment Search Tool) (Zhang et al., 2012). Multiple sequence alignments and construction of the phylogenetic trees were done using the neighbor-joining method using MEGA6 software (Tamura et al., 2013).

3. Results and discussion

3.1. Morphological and molecular identification of C. sphaerospermum isolates

C. sphaerospermum isolates were preliminary diagnosed based to the morphological characteristics. Colonies grown on PDA medium were dark olivaceous to greenish-black in color and velvety. The fungal conidiophores were closely septated with thick and darkened septa as well as branched. However, conidia were in branched-chain, small terminal conidia were apex rounded, brown to dark brown, globose to subglobose, base rounded to slightly attenuated each with a prominent scar at the end of the ramoconidia and conidium. The morphological characteristics of the isolates demonstrated that these fungal isolates belong to C. sphaerospermum. Bensch et al. (2018) reported that the main characteristics of C. sphaerospermum is the presence of numerous globose to subglobose terminal conidia, almost smooth to verruculose and conidiophores branched. Avasthi et al. (2016) and Yew et al. (2016) also stated similar results regarding morphological characteristics of C. sphaerospermum.

The morphological identification of the C. sphaerospermum isolates were also confirmed by PCR amplification of DNA extracted from these isolates. PCR amplification showed the PCR products with sizes ranging between 550-600bp by the universal primer pair (ITS1-ITS4) (Figure 1).

Figure 1
1% Agarose gel electrophoresis of PCR products amplified using the primer pair ITS1 and ITS4 from the C. sphaerospermum isolates (1-13) obtained from tomato plant residues collected from different regions of Najaf and Karbala provinces. M: 1Kbp DNA Ladder (Promega, USA).

The samples were sequenced in both directions and a BLAST search revealed that all the studied isolates belong to the fungus C. sphaerospermum. By comparing the partial sequence of ITS region of the C. sphaerospermum isolates (1-13) with the other C. sphaerospermum isolates previously available in GenBank, results showed that the similarity percentage of the nucleotide sequences ranged between 97%-100%. It was also noted that the nucleotide sequences of the ITS region (ITS1, 5.8S rDNA, and ITS4) amplified from the C. sphaerospermum isolates 5, 11, and 12, as well as 8 and 13, had a similarity of 100% (Table 2, Figure 2 and Figure 3). Comparison of the partial ITS region of the C. sphaerospermum isolates 1, 3, 4, 5, 7, 8, 11, 12, and 13 with formerly registered isolates in GenBank showed that the nearest nucleotide sequence similarity (100%) with C. sphaerospermum already isolated and identified in Japan (AB572902), Brazil (MK913353), China (MK356737), China (MK281560), China (MK356729), Iran (KY046241), China (MK281560), China (MK281560), and Iran (KY046241), respectively.

Table 2
Similarity rates among the C. sphaerospermum isolates identified in this study.

													Isolate no.
												-	1
											-	98	2
										-	99	99	3
									-	99	98	100	4
								-	99	100	98	99	5
							-	98	99	98	97	97	6
						-	99	99	100	99	98	100	7
					-	100	99	99	100	99	98	100	8
				-	99	99	98	98	98	99	99	97	9
			-	98	99	99	98	98	99	99	97	99	10
		-	98	98	99	99	98	100	99	100	98	99	11
	-	100	98	98	99	99	98	100	99	100	98	99	12
-	99	99	99	99	100	100	99	100	99	98	98	100	13
13	12	11	10	9	8	7	6	5	4	3	2	1

Figure 2
The similarity and difference in the nucleotide sequences of the C. sphaerospermum isolates (1-13) identified in the present study. Similar nucleotides are stated in dots. Numbers given on the right side of the figure represent the nucleotide sequences obtained from the C. sphaerospermum isolates.

Figure 3
A phylogenetic tree constructed by the neighbor-joining method depending on a comparison of the nucleotide sequences obtained from the C. sphaerospermum isolates (1-13).

Analysis of the C. sphaerospermum isolate 2 showed distinct differences in some nucleotide positions with the C. sphaerospermum isolates previously identified and registered in GenBank (Figure 2). This isolate (2) had a 98%-99% nucleotide sequence similarity with the other isolates available at NCBI (Figure 3).

A comparison of the nucleotide sequence obtained from this isolate (2) with those recorded in GenBank revealed that the highest similarity was 99% with the C. sphaerospermum isolates previously diagnosed in China (MK952405 and KU350727). Whereas, minimum nucleotide sequence similarity (98%) for this C. sphaerospermum isolate was observed with the isolates already identified in China (MG787259), Japan (LC317549, LC317550, LC375369, and LC317551), and Germany (MH482916) (Figure 4). The constructed phylogenetic tree showed that the C. sphaerospermum isolate 2 appeared in a separate clade from C. sphaerospermum isolates previously identified and recorded in GenBank (Figure 5).

Figure 4
The similarity and difference of nucleotide sequences in the C. sphaerospermum isolate 2, identified in this study, with those C. sphaerospermum isolates previously registered in NCBI. Identical nucleotides are given in dots.

Figure 5
A phylogenetic tree was generated using the neighbor-joining method which shows the genetic relationship between C. sphaerospermum 2 (as indicated in red circle) and the other C. sphaerospermum isolates deposited in GenBank (NCBI)

By comparing the nucleotide sequence obtained from the C. sphaerospermum isolate 6 with the other isolates registered in NCBI indicated that there was a clear similarity ranges between 95%-98% (Figure 6). The nearest sequence similarity was 98% with the C. sphaerospermum isolates already isolated in China (GQ901954), India (MF380926), and Iran (Iran). In contrast, the lowest homology percentage of 95% was noticed with the other isolates of C. sphaerospermum identified in China, Australia, the USA, as well as New Zealand. To determine the genetic relationship among these, the sequence of the C. sphaerospermum isolate (6) with the sequences of those C. sphaerospermum isolates available in GenBank were used for constructing a phylogenetic tree. The phylogenetic tree showed the appearance of this isolate 6 in a separate clade apart from the C. sphaerospermum isolates deposited in GenBank (Figure 7).

Figure 6
Nucleotide sequence alignment of the ITS region of the C. sphaerospermum isolate 6 identified in the current study and the other isolates already recorded in NCBI.

Figure 7
The Neighbor-Joining tree analysis shows the genetic relationship of the C. sphaerospermum isolate 6, investigated in this study, and the other isolates already recorded in NCBI.

As shown in Figure 8 and Figure 9, the C. sphaerospermum isolate 9 investigated in this study had clear differences in the nucleotide sequences ranged from 97%-99% with the C. sphaerospermum isolates previously registered in NCBI. C. sphaerospermum 9 revealed that the highest nucleotide sequence similarity was 99% with the C. sphaerospermum isolates already identified in Bangladesh (MH393182), Iran (KY046241), France (MH393182), and Australia (EF568045). Minimum similarity (97%) for this C. sphaerospermum isolate 9 was found with C. sphaerospermum isolates registered in Australia (KU059910), Chin (KP269061), and Netherlands (MF473024).

Figure 8
Nucleotide sequence alignment of the ITS (ITS1, 5.8S rDNA and ITS4) region of the C. sphaerospermum isolate 9 and the other isolates already recorded in NCBI.

Figure 9
A phylogenetic tree shows the genetic relationship between the C. sphaerospermum isolate 9 investigated in this study as indicated by red dote (●), and the C. sphaerospermum isolates available in NCBI.

A BLAST search was also done on the identified sequence of the C. sphaerospermum isolate 10 and analysis of nucleotide sequence alignment was carried out with the other sequences deposited in GenBank. Results showed that the sequence obtained from C. sphaerospermum isolated in the present study had a high degree of nucleotide identity (99%) with C. sphaerospermum isolates formerly isolated from Malaysia (JX966565 and JX966567), India (MF46882), Australia (EF568045) and China (KX885028 and KT852577). Whereas, the lower identity (98%) was revealed with C. sphaerospermum isolates previously identified in Japan (LC317550 and LC317551) (Figure 10). As illustrated in Figure 11, construction of the phylogenetic tree showed clustering of C. sphaerospermum 10 with the other C. sphaerospermum isolates already identified in Australia (EF568045), India (MF46882), and Malaysia (JX966565 and JX966567).

Figure 10
Nucleotide sequence alignment of the ITS (ITS1, 5.8S rDNA and ITS4) region of the C. sphaerospermum isolate 10 and the other isolates already recorded in NCBI.

Figure 11
A phylogenetic tree constructed using the neighbor-joining method depending on the comparison of the obtained nucleotide sequence of C. sphaerospermum isolate 10 as indicated by red dote (●) with those of C. sphaerospermum isolates deposited in NCBI.

The new C. sphaerospermum isolates identified in this study may be more devastating for some economic plants. In this study, PCR was successfully used to identify different isolates of C. sphaerospermum because of its high sensitivity and accuracy in the identification of many organisms, including fungi such as Fusarium oxysporium, Alternaria alternata, F. solani, Trichoderma spp., R. solani, and Aspergillus spp. (Al-Abedy et al., 2018; Khan et al., 2018).

Fungal identification has increased dramatically with the introduction of some molecular tools such as PCR. PCR and sequencing have widely invested as a quick and precise technique to identify many pathogenic and non-pathogenic plant fungi, bacteria, and viruses. Molecular markers have a role in eliminating the limitations in identification based on morphological characteristics (Henry et al., 2000; AL-Abedy et al., 2020). Despite this, morphological characters are being used in sorting fungal isolates into smaller groups but it requires time, efforts and expertise to be a taxonomist for accurate identification, particularly up to species level (Zhang et al., 2012; Huang et al., 2006; Abdullah et al., 2019). Morphological characteristic also depends on many other factors, e.g. moisture, pH, light, and composition of the growth medium that can change the color, shapes, and sizes of spores as well as the fungal colonies growing pattern (Zhang et al., 2012; Huang et al., 2006).

In Malaysia, studies on Fusarium spp. are often based on some morphological characters that could lead to incorrect identification (Hsuan et al., 2011). Previous studies revealed some limitations related to the use of morphological characters for the identification of some fungi, e.g. species in the G. fujikuroi species complex as some species, i.e., F. sacchari, F. subglutinans, F. verticillioides, F. proliferatum, F. fujikuroi, and F. andiyazi, have very close morphological characters (Hsuan et al., 2011). It was also noticed through the re-diagnosis by using the PCR technique that there was a mistake in the morphological identification of several fungi identified in previous studies like Fusarium spp., e.g., F. verticillioides, and Fusarium subglutinans (Hsuan et al., 2011).

Differences in the ITS (Internal Transcribed Spacer) regions of the rDNA (ribosomal DNA), repeat units are present in copies in fungi that can be found and selected by PCR (Al-Fadhal et al., 2018). PCR amplification of the ITS region has shown a high efficiency in identifying several fungi, e.g. Pythium spp., R. solani, and F. verticillioides (Hsuan et al., 2011; Al-Fadhal et al., 2018; AL-Abedy et al., 2018). It is concluded that the newly identified C. sphaerospermum isolates 2, 6, 9, and 10 were genetically different from the other C. sphaerospermum isolates and not previously recorded in NCBI; therefore, they were registered in GenBank under the accession numbers: MN896004, MN896107, MN896963, and MN896971, respectively.

Acknowledgements

The authors would like to thank the Head of Department of Plant Protection, University of Kerbala, for providing essential laboratory facilities and support to conduct this research work successfully.

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Publication Dates

• Publication in this collection
  09 July 2021
• Date of issue
  2022

History

• Received
  30 Apr 2020
• Accepted
  05 Nov 2020