Introduction

Pakistan is in top 15 Citrus producing countries and area covered by Citrus is 206569 ha producing 1907.4 thousand tonnes, 98% of which is produced by Punjab province only (FAO 2017). Area in Punjab with Citrus orchards is 183210 ha with 2135.895 thousand tones production (GOP 2016–2017). The most widely grown and used variety is kinnow (Citrus reticulata) which covers the 86% of the total area in Pakistan (Altaf 2006). Important districts for citrus cultivation are Sargodha, Sahiwal, Multan, Jhang, Lahore, Gujranwala, Mianwali and Sialkot.

Citrus withertip/dieback is serious damage causing disease in Pakistan. Symptoms are characterized by yellowing of the leaves and drooping of leaves occurs as wilting progressed leading to death of the plants. Symptoms start from the tip of the twigs and extend towards the main stem causing dieback (Al-Sadi et al. 2014). Black pustules were clearly observed on dead twigs and in severe cases silvery appearance was found on twigs. Citrus withertip was first time reported in Mexico by Benyahia et al. (2003) caused by Colletotrichum gloeosporioides. In Ghana, C. gloeosporioides was found to be associated with Cassava stem tip dieback disease (Moses et al. 1996). Eucalyptus dieback was also caused by C. gloeosporioides in South Africa (Smith et al. 1998). In Oman, Fusarium solani, Lasiodiplodia theobromae, Neoscytalidium dimidiatum and L. hormozganensis were discovered to be associated with Citrus on lime seedlings (Al-Sadi et al. 2014).

Lately, molecular techniques based on DNA have been developed for the identification and characterization of fungal species. In past, there are many studies in which phylogeny is inferred using internal transcribed spacer (ITS) region of rDNA to distinguish the complex fungal species (Cen et al. 1994; Moses et al. 1996; Smith et al. 1998) but using ITS sequences alone are skeptical to identify and characterize the closely related complex fungal species (Weir et al. 2012; Hassan et al. 2018). While the technology progressed, different methods have been adapted all over the world for identification and diversification of the species among the genera (Liu et al. 2016; Rodriguez-Galvez et al. 2017). ITS sequences alone cannot distinguish the Colletotrichum species. For C. gloeosporioides complex, six protein coding genes are used as secondary barcodes other than ITS region; actin (ACT), glutamine synthetase (GS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS) and beta-tubulin (βTUB) (Weir et al. 2012; Hassan et al. 2018).

Although, citrus withertip/dieback is an emerging disease, it has been neglected from many years. Despite the importance of this disease no cultural and molecular studies have been performed to identify the causal agent(s). It has become a necessity to fill this gap to understand the etiology of this disease which will enable us to adopt better management practices. Current study was designed to properly investigate the causal agent of citrus withertip/dieback in the orchards of Punjab, Pakistan and to become a milestone for future studies.

Materials and Methods

Samples collection

In 2015–2016, samples were obtained from orchards of four major citrus growing areas of Punjab province (Sargodha, Kot Momin, Bhalwal and Faisalabad) from plants showing characteristic symptoms of withertip/dieback (Fig. 1). Samples were obtained from two orchards of each district. The diseased samples were collected from trees of cultivar Kinnow (C. reticulata). The diseased samples were placed in paper bags with labels, were brought to Molecular Plant Pathology laboratory, University of Agriculture, Faisalabad (UAF), Pakistan and placed in refrigerator at 4°C for further use.

Morphological identification and pathogenicity analysis

Diseased portion from the edges of lesions were cut into 2–3 mm size pieces and used for isolation of pathogen. Samples were disinfected with 70% ethanol for one minute and with distilled water again. These samples were placed aseptically onto Potato Dextrose Agar (PDA) media and incubated at 30–35°C in N-BIOTEK incubator (NB-205LF) for 6 days and observe daily for growth. Sub culturing was done from mycelium on PDA in the test tubes (30 mL cap.) to get pure culture and then stored in refrigerator at 4°C. Different morphological characters;

Fig. 1: Trees showing characteristics symptoms of withertip/dieback: A; progressed dieback no fruit formation, B; onset of dieback, C; Leaf chlorosis

colony colour and growth, conidia size and fruiting bodies were observed.

Pathogenicity trials were done by using Koch’s postulates in green house. One-year old kinnow plants were transferred in pots (9² (diameter) x 8.5² (depth)) filled using aseptic soil. For preparation of spore suspension, the protocol of Mello et al. (2004) was followed. The counting of spores was done with hemocytometer and 10⁴/mL spore suspension was inoculated on healthy plants by spraying. After the appearance of symptoms, re-isolation was done following the Koch’s postulates.

Whole genome sequencing and phylogenetic analysis

Fungal DNA was extracted by modified CTAB method (Moller et al. 1992) from pure fungal culture. Pellet were dissolved in 25 μL of sterile distilled water (SDW) and stored at -20°C. Extracted DNA was used for the preparation of sequencing libraries at the Cook’s Lab, University of California, Davis, USA, following Qiagen® QIAseq FX ®DNA Library Kit protocol. Illumina HiSeq 4000 sequencing was performed at the Genome Center, University of California, Davis, USA. Assembly of genomes was done by using SPAdes 3.9.0 pipeline (Bankevich et al. 2012). Assemblies were produced using ‘careful’ mode in SPAdes to avoid miss-pairing of contigs by scaffolding. Phylogenetic relationship was determined by ML method of MEGA 7.0 with 1000 replicate of six genes (ACT, CAL, CHS1, GAPDH, GS, TUB2) along with ITS to identify the species of Colletotrichum. Average nucleotide identity (ANI) was measured by using Pyani (Pritchard et al. 2016).

Results

Morphological characters

After isolation and purification, two different groups of fungi were identified belonging to C. gloeosporioides complex. One group resembles with C. gloeosporioides; Grey-orange color colony growth with concentric rings was observed on the opposite side of the petri dish. Wooly thick mycelium was hyaline and septate. Black conidiophores were also observed and conidia were hyaline with size of 15–20 × 2.5–5 mm, cylindrical with

Fig. 2: Morphological characteristics of Colletotrichum-Group-a; A: Orange grey mycelial growth of representative isolate of the group, B: Black colony growth after 10 days, C: opposite side of the petri dish showed orange circular growth, D: Ruptured conidiophores, E: conidia and mycelium under microscope at 20X

obtuse base and rounded apex (Fig. 2). Other groups members resemble with C. siamense; White colored colony growth observed which later turn into grey color with scattered light orange acervuli. Mycelium was hyaline and septate. Conidia were hyaline with size of 12.5–20 × 3.5–5.5 mm, cylindrical to fusiform in shape and aseptate (Fig. 3).

Pathogenicity assay

Pathogenicity trial on one-year old Kinnow plants confirmed that causal agents of the citrus withertip/dieback are C. siamense and C. gloeosporioides. Onset of disease was recorded after 10 days of inoculation from tips of the plants following leaf chlorosis, defoliation and dieback. Plants died after four weeks of inoculation. Characteristic symptoms were recorded during the pathogenicity trial (Fig. 4). Re-isolation of the fungi was performed successfully to confirm the Koch’s postulates from disease seedlings.

Molecular identification and phylogenetic analyses

Fig. 3: Morphological characteristics of Colletotrichum-Group-b; A: White mycelial growth of representative isolate of the group, B: Colony growth after 14 days showed scattered orange and black conidiophore, C: fruiting bodies along with mycelium, D: conidia and mycelium under microscope (20X), E: At 40X showing granular structure of conidia

Fig. 4: Pathogenicity Trail on one-year old Citrus plants; A: Onset of disease; B: Plant died after four weeks post-inoculation

Genome size of C. siamense strains were ~56 Mbp and C. gloeosporioides were of 56–61 Mbp. The assembled genomes were deposited to NCBI and accession number were assigned to respective genomes (Table 1). Six genes (ACT, CAL, CHS1, GAPDH, GS, TUB2) along with ITS were extracted, concatenated and used to identify and to inferred the phylogeny of different Colletotrichum species presented in Table 2. The Table 1: Pakistani strains isolated from different district of Punjab

Strains	No. of scaffolds	Longest fragment	Shortest fragment	Genome size	Rate of N	Rate of GC	Scaffold N50	No. of sequences >=3 kb	Accession Number
COLG-31	4142	149620	128	56492417	1.07E-05	0.52263813	25160	3084	VNWS00000000
COLG-34	17516	100753	128	61427343	4.23E-06	0.52996204	16081	4046	WEZJ00000000
COLG-38	8793	261654	128	59174470	3.89E-06	0.53030821	38314	2165	WEZK00000000
COLG-44	6278	115451	128	58427301	1.49E-05	0.52538398	16594	4337	WEZL00000000
COLG-50	6784	80160	128	56927278	8.78E-07	0.52498492	16038	4281	WEZM00000000
COLG-90	5456	279223	128	57669018	1.73E-06	0.52938951	48402	1875	WEZN00000000
COLG-95	5237	373335	60	56028021	1.03E-05	0.52332074	20160	3635	WEZO00000000

Table 2: The list of fungal genomes used for comparative analysis

Isolates Used in the analysis	Strain name	Gene Bank Assembly Accession No.	Host	Origin
C. fructicola	1104-7	GCA_002314275.1	Apple	China
C. fructicola	15060	GCA_002887685.1	Mango	China
C. fructicola	Nara gc5	GCA_000319635.1	Fragaria x ananassa	Japan
C. gloeosporoides	030206	GCA_002189585.1	Apple	China
C. gloeosporoides	cg-14	GCA_000446055.1	Avocado cv. Fuente	Israel
C. gloeosporoides	cg01	GCA_003666125.1	Huperzia serrata	China
C. gloeosporoides	ES026	GCA_003568745.1	H. serrata	China
C. gloeosporoides	TYU	GCA_002901105.1	Taxus cuspidata	South Korea
C. incanum	MAFF238704	GCA_001625285.1	Raphanus sativus L.	Japan
C. incanum	MAFF238712	GCA_001855235.1	R. sativus var. longipinnatus	Japan
C. graminicola	M1001	GCA_000149035.1	N/A	N/A
C. graminicola	M5	GCA_001951205.1	Zea mays	Brazil
C. sublineola	CgSl1	GCA_001951195.1	Sorghum bicolor	USA
C. sublineola	TX430BB	GCA_000696135.1	S. bicolor	USA
C. higginsianum	IMI 349063	GCA_001672515.2	Brassica rapa subspp. chinensis	Trinidad & Tobago
C. coccodes	NJ-RT1	GCA_002249775.1	pepper fruit	USA
C. coccodes	RP180a	GCA_002249805.1	pepper fruit	USA
C. tofieldiae	0861	GCA_001625265.1	Arabidopsis thaliana	Spain
C. tofieldiae	CBS 127615	GCA_001618715.1	Agapanthus spp.	Portugal
C. tofieldiae	CBS 168.49	GCA_001618705.1	Lupinus polyphyllus	Germany
C. tofieldiae	CBS 495.85	GCA_001618725.1	Tofieldia calyculata	Switzerland
C. tofieldiae	CBS 130851	GCA_001618735.1	Semele androgyna	Germany
C. fioriniae	HC89	GCA_002930455.1	Apple	USA
C. fioriniae	PJ7	GCA_000582985.1	Fragaria x ananassa	New Zealand
C. fioriniae	HC91	GCA_002930425.1	N/A	N/A
C. acutatum	C71	GCA_001662755.1	N/A	N/A
C. acutatum	1	GCA_001593745.1	Capsicum annuum	Korea
C. nymphaeae	SA-01	GCA_001563115.1	N/A	N/A
C. lindemuthianum	89 A2 2-3	GCA_001693025.2	Phaseolus vulgaris	USA
C. lindemuthianum	83.501	GCA_001693015.2	P. vulgaris	USA
C. salicis	CBS 607.94	GCA_001563125.1	Salix spp.	Netherlands
C. spinosum	CBS 515.97	GCA_004366825.1	N/A	N/A
C. simmondsii	CBS122122	GCA_001563135.1	Papaya	Australia
C. tanaceti	BRIP57314	GCA_005350895.1	Tanacetum cinerariifolium	Australia
C. sansevieriae	Sa-1-2	GCA_002749775.1	N/A	Japan
Colletotrichum spp.	JS-367	GCA_003122705.1	Mulberry	South Korea
C. gloeosporioides	SMCG1	GCA_003243855.1	Chinese fir	China
C. chlorophyti	NTL11	GCA_001937105.1	N/A	N/A
C. lentis	CT-30	GCA_003386485.1	Lens culinaris sspp. culinaris	Canada
C. orbiculare	104-T	GCA_000350065.2	Cucumber	N/A
C. sidae	CBS 518.97	GCA_004367935.1	N/A	N/A
C. truncatum	MTCC 3414	GCA_002632455.2	Capsicum annuum	India
C. trifolii	543-2	GCA_004367215.1	N/A	N/A
C. falcatum	Cf671	GCA_001484525.1	Sugarcane	India
C. musae	GM20	GCA_002814275.1	N/A	N/A
C. higginsianum	MAFF305635	GCA_004920355.1	N/A	N/A
C. graminicola	M5	GCA_001951205.1	Maize	Brazil
Colletotrichum spp.	PG-2018a	GCA_006783085.1	Perilla	N/A

inferred phylogeny revealed two Colletotrichum species. COLG-31 and COLG-95 had affinity to C. gloeosporioides_IMI-356878 with a bootstrap value of 100%. While COLG-34, COLG-38, COLG-44, COLG-50 and COLG-90 grouped with different C. siamense isolates, also with strong bootstrap support (Fig. 5). Another method of pairwise comparison was used to determine the specie relationship using Average nucleotide identity (ANI) of 95%. ANI₉₅ also supported the phylogenetic analysis dividing the isolates into two different groups. COLG-31 and COLG-95 grouped with C. gloeosporioides with high ANI value (98%) along with high pairwise genome alignment coverage (92–96%), supporting strong relationship with C. gloeosporioides. Strains COLG-34, COLG-38, COLG-44, COLG-50 and COLG-90 formed individual group with 99% ANI and 92–94% genome alignment coverage (Fig. 6). Phylogenetic analysis revealed close affinity with C. siamense, however no genome for C. siamense was available in public databases to compare with before June 2019.

Discussion

Fig. 5: Phylogenetic analysis using ITS, ACT, CAL, CHS1, GAPDH, GS, TUB2 genes of Pakistani isolates with Publicly available strains from NCBI by ML method

Fig. 6: Average Nucleotide identity (ANI) Analysis all the genomes available on NCBI along with Pakistani isolates dividing into two distinguish groups; C. siamense and C. gloeosporioides

Citrus withertip/dieback has been prevailing in the Pakistan from decades; however, the pathogens associated with this disease have never been identified and characterized on molecular basis. For this purpose, the study was designed to identify and characterize the pathogens associated with citrus withertip/dieback which ultimately will help in better understanding of the disease and its management. In 2015, samples were collected from different orchards of Punjab; dieback symptoms were observed resulting considerable yield losses following the complete plant death. The recent discovery of several new species of fungi associated with tropical plants led us to speculate that more than one fungus is associated with dieback of Citrus in Pakistan. In vitro morphological characters are important for distinguishing among Colletotrichum species (Hu et al. 2015) and on this basis the isolated fungi were further divided into 2 sub-groups. The identified isolates of C. gloeosporioides and C. siamense showed distinct colony morphologies, with small variations in appressoria, conidia sizes and shape, which were similar to morphological characters of C. gloeosporioides (Kimaru et al. 2018) and C. siamense (Cristóbal-Martínez et al. 2017) (Fig. 2 and 3). All of the tested Colletotrichum isolates showed similar symptoms and degree of virulence when tested for Koch’s postulates. For molecular characterization of isolated pathogenic fungi, high quality draft genomes were produced to check the gene contents of both species and to understand the host-pathogen interaction. Since ITS region is not enough to identify the Colletotrichum species (Weir et al. 2012), so six different genes along with ITS (ACT, CAL, CHS1, GAPDH, GS, TUB2) was used to identify different species in given isolates. However, in previous research only C. gloeosporioides and C. theobromicola were reported to cause dieback (Smith et al. 1998; Singh et al. 2015; Hawk et al. 2018). C. siamense is first time reported to cause dieback on citrus along with C. gloeosporioides. ANI₉₅ has been used widely to differentiate the isolates on specie level (Goris et al. 2007; Richter and Rosselló-Móra 2009). Average nucleotide identity (ANI) analysis and phylogenetics analysis clearly distinct the isolates belonging to two different species within the Musae clade. Until now, F. solani, L. hormozganensis, N. dimidiatum and L. theobromae is found to be associated with Citrus Dieback (Ferrari et al. 1996; Al-Sadi et al. 2014). But present studies revealed that C. gloeosporioides and C. siamense for citrus dieback/withertip in Punjab, Pakistan. These findings led to the better understanding of the disease and can help for further analysis to establish management strategies for Citrus withertip/ dieback. In Future, these isolates may play a fundamental part in refining our understanding to the extent of cryptic species diversity in Colletotrichum complexes.

Conclusion

We identified two species from C. gloeosporioides complex that caused dieback on citrus, based on morphological, pathogenesis, and molecular analyses. The identification of a new Colletotrichum spp. causing dieback on citrus redirects the importance of further research on Colletotrichum taxonomy to alleviate the risk to the citrus fruit industry not only in Pakistan but throughout the world.

Acknowledgements

This research was carried out as a part of grant project 20-2789 NPRU supported by Higher Education Commission and IRSIP funding. The financial support is greatly appreciated. We would also like to thank The Ohio State University, for providing Supercomputer resources.

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