Journal of Plant Pathology

Morphological ambiguity in Lasiodiplodia theobromae (Pat.), an emerging pathogen

causing dieback disease in Pelargonium graveolens L'Her, in northern India
--Manuscript Draft--

Manuscript Number: JPPY-D-23-00811

Full Title: Morphological ambiguity in Lasiodiplodia theobromae (Pat.), an emerging pathogen

causing dieback disease in Pelargonium graveolens L'Her, in northern India

Article Type: Original Paper

Funding Information: Central Institute of Medicinal and Aromatic Dr Kishore Babu Bandamaravuri
Plants
(Aroma Mission-HCP-007)

Abstract: Pelargonium graveolens, commonly known as rose scented geranium, is an important

aromatic and medicinal shrub from the Geraniaceae family. The aerial parts of the
plant contain geraniol and citronellol, which are traditionally used in medicine, insect
repellents, perfumes, and flavoring. Over three consecutive years, surveys were
conducted during the early rainy season in geranium fields, revealing severe dieback
symptoms with disease incidence ranging from 54% –78%. The pathogen isolated from
infected plant tissue exhibited greyish brown to blackish grey dense aerial mycelia. The
cultural as well as the conidial morphology of the pathogen isolate closely resembled
that of L. theobroemae, the dimensions of the conidia in this study differed (10-15×3-
7μm) from the earlier reported size (27-35×15-20 μm). To clear this ambiguity, a
detailed molecular analysis was carried out.The analysis of combined results of
microscopy as well as molecular data showed highest similarity with L. theobroemae,
thus confirming the association of pathogen L. theobromae to dieback disease of the
rose-scented geranium crop.

Corresponding Author: Kishore Babu Bandamaravuri, Ph.D

CSIR-CIMAP: Central Institute of Medicinal and Aromatic Plants CSIR
Lucknow, INDIA

Corresponding Author Secondary

Information:

Corresponding Author's Institution: CSIR-CIMAP: Central Institute of Medicinal and Aromatic Plants CSIR

Corresponding Author's Secondary

Institution:

First Author: Bhanu Sharma, MSc

First Author Secondary Information:

Order of Authors: Bhanu Sharma, MSc

A Kumar, MSc

Saudan Singh, PhD

Kishore Babu Bandamaravuri, Ph.D

Order of Authors Secondary Information:

Author Comments: Highlights of the manuscript include:

- The survey results in this research shed light on the devastating consequences of
dieback infection, that include huge crop losses and in turn massive loss of money to
the farmers.
- Comparative analysis of conidia sizes in the genus Lasiodiplodia with the sizes
obtained in the microscopic study in the current study created an ambiguity in species
level characterization. In order to find a solution to it, a detailed molecular and
phylogenetic analysis has been carried out, with multilocus gene sequences in multiple
sequence alignment with all the known species of the genus.
- This research establishes the fungus L. theobroemae as the pathogen causing
dieback in P. graveolens.
The potential implications of this research extend beyond academia, with implications

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
 for agricultural practices and crop protection.

Suggested Reviewers: Lakshman Prasad, PhD

Principal Scientist, Indian Agricultural Research Institute
lakshmanprasad25@yahoo.com
He is involved in research related to fungal plant pathology.

Alves Artur, PhD

Assistant Professor, University of Aveiro: Universidade de Aveiro
artur.alves@ua.pt
He is a mycologist/ microbiologist working on microbial diversity and evolution

Selvarajan R., PhD

Director, NRCB: National Research Centre for Banana
selvarajanr@gmail.com
He is involved in research related to plant pathology

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation
Cover letter Click here to access/download;Cover letter;Cover letter.doc

To

The Editor-in-Chief

Journal of Plant Pathology

Dear Dr. Garbelotto,

I am writing to submit my research paper entitled "Morphological ambiguity in Lasiodiplodia

theobromae (Pat.), an emerging pathogen causing dieback disease in Pelargonium graveolens
L'Her, in northern India" to be considered for publication in Journal of Plant Pathology. The
potential implications of this research extend beyond academia, with implications for
agricultural practices and crop protection, thus, it holds significant value and relevance to the
readership of your esteemed journal. All authors have read and agree to the submission and
that the study has not been submitted elsewhere for publications.

Desired Senior Editor and/or Associate Editor to handle the submission- Luana Giordano,
Laboratory of Lombardy Plant Health Service, Fondazione Minoprio, Vertemate con
Minoprio, Italy

In this paper, we have conducted an in-depth study to characterize the causative fungus of
dieback disease in P. graveolens. This research is based on robust experimental design and
extensive data analysis. Highlights of the manuscript include:

- The survey results in this research shed light on the devastating consequences of dieback
infection, that include huge crop losses and in turn massive loss of money to the farmers.

- Comparative analysis of conidia sizes in the genus Lasiodiplodia with the sizes obtained
in the microscopic study in the current study created an ambiguity in species level
characterization. In order to find a solution to it, a detailed molecular and phylogenetic
analysis has been carried out, with multilocus gene sequences in multiple sequence
alignment with all the known species of the genus.

- This research establishes the fungus L. theobroemae as the pathogen causing dieback in
P. graveolens.

List of possible reviewers for this paper-

a) Dr. R. Selvarajan - (ICAR-National Research Centre For Banana, India;

selvarajanr@icar.org.in )- He is involved in research related to plant pathology
b) Dr. Artur Alves- (University of Aveiro, Portugal; artur.alves@ua.pt )- He is a
mycologist/ microbiologist interested in microbial diversity and evolution

c) Dr Andy M Bailey- (University of Bristol, UK; Andy.Bailey@bristol.ac.uk )- He has

long been involved in research on fungal molecular genetics and plant pathology
I eagerly anticipate your response and look forward to the opportunity to contribute to the
scientific community through your journal.

Thank you for your consideration.

Regards.

Dr. Kishore B. Bandamaravuri

Principal Scientist

CSIR-CIMAP, Lucknow

INDIA
Manuscript Click here to access/download;Manuscript;Artwork-PgDbK- J.
of Plant Pathology.doc
Click here to view linked References

Morphological ambiguity in Lasiodiplodia theobromae (Pat.), an emerging pathogen

1
2 causing dieback disease in Pelargonium graveolens L'Her, in northern India
3
4
5 Bhanu Sharma1, Anuj Kumar1, Saudan Singh1 and Kishore Babu Bandamaravuri1*
6
7
8 1- Crop Production and Protection Division, CSIR-Central Institute of Medicinal and Aromatic Plants,
9 Lucknow, 226015, India
10
11
12 *Corresponding author email: kishorebanadam@cimap.res.in
13
14
15 Abstract
16
17
Pelargonium graveolens, commonly known as rose scented geranium, is an important aromatic and medicinal
18
19 shrub from the Geraniaceae family. The aerial parts of the plant contain geraniol and citronellol, which are
20
21 traditionally used in medicine, insect repellents, perfumes, and flavoring. Over three consecutive years, surveys
22 were conducted during the early rainy season in geranium fields, revealing severe dieback symptoms with
23
24 disease incidence ranging from 54% –78%. The pathogen isolated from infected plant tissue exhibited greyish
25 brown to blackish grey dense aerial mycelia. The cultural as well as the conidial morphology of the pathogen
26
27 isolate closely resembled that of L. theobroemae, the dimensions of the conidia in this study differed (10-15×3-
28 7μm) from the earlier reported size (27-35×15-20 μm). To clear this ambiguity, a detailed molecular analysis
29
30 was carried out. The analysis of combined results of microscopy as well as molecular data showed highest
31 similarity with L. theobroemae, thus confirming the association of pathogen L. theobromae to dieback disease
32
33 of the rose-scented geranium crop.
34
35
Keywords: disease incidence, geraniole, phytopathogen, rose scented geranium
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 Page 1 of 15
63
64
65
 Introduction
1
2
3 Pelargonium graveolens L'Her, commonly known as rose scented geranium or geranium is a member of the
4 Geraniaceae family, native of South Africa, and also found widely in China, Egypt, Morocco, Russia, Algeria,
5
6 Israel, and India. As a perennial crop, P. graveolens is commercially cultivated for essential oil production in
7 temperate and sub-tropical climate (Rajeswara et al. 2000b). The essential oil is frequently used by the perfume
8
9 and cosmetics industries (Harborne et al. 2002, Ben Hsouna et al. 2012). Researchers estimate that the global
10 production of geranium oil reaches approximately 400 metric tons per year, and it holds a market value of $20-
11
12 30 million USD (Pandey et al. 2020).
13
14
15 Several studies indicate that geranium essential oil components have antioxidant, antibacterial, antifungal,
16 antiviral, and antiseptic properties (Androutsopoulou et al. 2021; Rajeswara 2009) and the essential oil is also
17
18 used in therapeutics for wound healing, ulcer, skin disorder, diarrhoea, dysentery, dermatitis, and as an anti-
19 diabetic, and anti-hemorrhoids (Boukhris et al. 2012). The distillate of fresh foliage, which has a distinctive
20
21 rose-like scent, is used as an alternative to rose oil (Blerot et al. 2016). According to reports, the leaves of rose-
22 scented geranium synthesise and accumulate tartaric acid, used as a food additive, in leavening, and as a flavour
23
24 enhancer (Narnoliya et al. 2019).
25
26
In India, geranium cultivation was initially introduced in the Nilgiri hills in Tamil Nadu. Since then, it has
27
28 spread to neighbouring states including Karnataka, Andhra Pradesh and in the north to Uttar Pradesh,
29
Uttarakhand, and Himachal Pradesh (Hurrah et al. 2021). Geranium is mostly cultivated in rainfed conditions in
30
31 the high-altitude hilly areas of the Himalayan regions, and irrigated conditions in the plains (Rajeswara et al.
32
33 1999; Wagh et al. 2020).
34
35 Inadequate farming practices employed in traditional cultivation, coupled with the delicate nature of the crop,
36
37 causes substantial losses to occur every season (Shawl et al. 2006). Significant losses are reported every season
38 due to attack of Rhizoctonia solani and Fusarium oxysporum causing root rot-wilt complex diseases (Prasad et
39
40 al. 2010). Fusarium wilt in plains results in severe yield losses (73.6-88.2%) and plant mortalities (70-80%).
41
Little leaf disease caused by phytoplasma or mycoplasma has also been detected in this crop (Rajeswara et al.
42
43 2000a). Prevalent damping-off infections on commercially grown geraniums are observed in the USA, Southern
44
states of England and other regions of the world. Pathogens associated with these infections are Pythium
45
46 ultimum, P. irregular, Rhizoctonia spp., Botrytis cinerea and Sclerotinia sclerotiorum (Munera et al. 2016; Zhao
47
et al. 2019).
48
49
50 In tropical and subtropical regions several species of Lasiodiplodia genus have been reported as serious plant
51
52 pathogens causing significant damages to the crops such as L. brasiliensis on watermelon (Netto et al. 2014), L.
53 euphorbiaceicola on Jatropha curcas (Machado et al. 2014), L. mediterranea on Vitis vinifera (Linaldeddu et al
54
55 2015). Another most significant plant pathogen falling under this genus is L. theobromae (Pat.), also referred to
56 as Botryodiplodia theobromae in many earlier records, has been linked to significant disease occurrences in
57
58 India (81 records) and Brazil (32 records) (Vardhana et al. 2017; Salvatore et al. 2020).
59
60
61
62 Page 2 of 15
63
64
65
 This fungus exhibits multiple modes of life cycles, as a pathogen (Abdollahzadeh et al. 2010; Dissanayake et al.
1 2015), an endophyte (Chen et al. 2015), and a saprophyte (Liu et al. 2012) on a wide range of hosts, with more
2
3 than 500 host plant species documented (Punithalingam 1980; Burgess et al. 2006; Dissanayake et al. 2016). L.
4 theobromae affects several economically important hosts, including mango dieback and fruit rot (Ismail et al.
5
6 2012), dieback of grapevine (Felix C et al. 2019), walnut soft rot and canker, postharvest damages in citrus
7 (Guajardo et al. 2018), off-shoot rot of Phoenix dactylifera (El-Ganainy et al. 2022), Ficus carica (Chen et al.
8
9 2018), root rot in beetroot, rot of papaya (Li et al. 2020), and Magnolia grandiflora (de Silva et al. 2019).
10
11
12 The rose scented geranium crop is found to be vulnerable to various diseases including stem rot, leaf blight,
13 wilt, and root rot caused by both fungal and bacterial pathogens particularly during rainy seasons (Rajeswara et
14
15 al. 2000a). In the current study, surveys were conducted in geranium cultivating fields and nurseries during the
16 monsoon season to document the prevalence of dieback disease in P. graveolens and provide a detailed analysis
17
18 of the pathogen responsible for its occurrence.
19
20
21 Materials and Methods
22
23
Disease survey and pathogen isolation from infected plants
24
25
26 During the monsoon seasons of 2017, 2018, and 2019, instances of natural dieback infections were observed on
27
28 previously healthy geranium plants and nursery blocks. A comprehensive survey was conducted in 52 fields
29 across six districts of Uttar Pradesh, India, as detailed in Table 1. Infected plant tissue samples were collected
30
31 and processed following the methodology described by Nishad et al. (2018) to isolate the causal agents. The
32 isolation process was carried out in all three years resulting in four isolates. After isolation, culture plates were
33
34 placed in a dark environment and incubated at a temperature of 27±2°C for a period of 5-7 days. The fungal
35 cultures were subsequently purified and maintained on PDA plates for further use.
36
37
38 Morphology and cultural characteristics of the fungi
39
40
41 The fungal isolates were subjected for morphological and microscopic characterization. Hyphal and spore
42 solutions were stained using lactophenol cotton blue, and observations were made using a light microscope
43
44 (DM750, Leica, Germany) at magnifications of 40× and 100×. The culture morphology and spore dimensions
45
were recorded and compared. As per the similar characteristics obtained in all the isolates, PgDbK03 was
46
47 selected for further studies.
48
49
50 Pathogenicity assay
51
52
To assess the pathogenicity of the representative isolate PgDbK03, an assay was conducted using healthy
53
54 geranium plantlets that were 15-20 days old and obtained through vegetative propagation. Spore suspension
55
(1x105 spores/ml) from 10 days old fungal cultures was prepared and spray inoculated onto the leaf surfaces of
56
57 the geranium plantlets, and the control plants were sprayed with sterile distilled water (SDW) instead (Nayak et
58
59 al. 2019). To ensure proper conditions for disease development, the control and inoculated plants were kept in
60
61
62 Page 3 of 15
63
64
65
 separate chambers and maintained at 27±2°C with a relative humidity of over 70% under glasshouse. The plants
1 were closely monitored for dieback symptoms over a period of 15-20 days after inoculation.
2
3
4 DNA extraction, PCR amplification and sequencing
5
6
7 The genomic DNA of the fungal isolates was extracted using the CTAB method and amplification and
8 sequencing of the ITS region of the ribosomal DNA (rDNA) cluster was performed as described by Nayak et al.
9
10 (2019). Amplification and sequencing of the translation elongation factor-1α (tef1-α) gene, was performed using
11 983F and 2218R primers (Alves et al. 2008). The obtained sequences were deposited to the NCBI GenBank
12
13 database.
14
15
16 Sequence alignment and phylogenetic analysis
17
18
Using BLAST analysis, reference sequences of Lasiodiplodia species were selected and downloaded from
19
20 GenBank (Table 2), for the purpose of phylogenetic analysis. Sequence alignment was carried out using
21
MEGAX (Kumar et al. 2018) and the MAFFT v7.307 online tool (https://mafft.cbrc.jp/alignment/server/). Any
22
23 ambiguous sequences and gaps were manually adjusted to optimize the alignments. The concatenated and
24
aligned sequence data comprised of 64 nucleotide sequences with a total of 1032 positions of ITS and tef1-α
25
26 regions. Phylogenetic tree construction was done using the Maximum Likelihood method and the Hasegawa-
27
28 Kishino-Yano model analysis. To assess the robustness of the tree, 1000 bootstrap replications were performed.
29 Diplodia mutila (3-1-51A) and Diplodia corticola (CBS 112073) were chosen for out-grouping. These outgroup
30
31 provided a reference point outside the Lasiodiplodia genus for the phylogenetic analysis.
32
33
Results
34
35
36 Disease Survey and pathogen isolation
37
38
39 The surveys were carried out over three consecutive years in geranium fields affected by disease in the districts
40
41 of Badaun, Sambal, Rampur, Barabanki, Sitapur, and Shahjahanpur. Popularly cultivated, cv. Bourbon and var.
42 CIM Pawan of geranium, were the focus of this study. The symptoms of dieback disease were clearly observed
43
44 on the plantlets (Fig 1). Initially, the disease symptoms manifested as yellowing of leaves and shoots, which
45 eventually turned black. In the advanced stages, chlorotic spots appeared on the middle stem, accompanied by
46
47 defoliation. These distinct symptoms spread across the field during favourable conditions such as high humidity
48 (>85%) in the rainy season and further the plants in the affected area succumbed to the disease, leading to
49
50 complete crop loss.
51
52
For the cv. Bourbon, the average incidence of the dieback disease over the course of three years ranged from
53
54 59.33% to 74.66%. In the case of var. CIM Pawan, the average disease incidences ranged from 56.66% to
55
69.66% across the same three-year seasons (Table 1). Similar symptoms and disease progression was observed
56
57 in all three years. In early July, the plants exhibited mild incidences, and as the season progressed, the severity
58
59 of the disease increased, and higher incidences were noted in late August. This indicates that the dieback disease
60 became more pronounced and prevalent as the rainy season advanced.
61
62 Page 4 of 15
63
64
65
 Pathogen isolation from the infected plant tissue samples resulted in four isolates such as PgDbK02, PgDbK03,
1 and PgDbK04, PgDbK05, isolated during the years 2017, 2018 and 2019 respectively.
2
3
4 Morphology and cultural characteristics of the fungi
5
6
7 The fungal colony on PDA plate exhibited both aerial and submerged growth, characterized by greyish brown to
8 blackish grey dense mycelia in the aerial part. Microscopic observations revealed that the fungus was spore
9
10 forming, branched, septate and coenocytic in nature. The hyphae of the fungus were branched, septate,
11 cylindrical and slightly curved. The sterile filaments called paraphyses, were observed to be cylindrical and
12
13 needle-shaped. They were hyaline and typically had 1-2 septa. The conidiogenous cells were holoblastic. They
14
were cylindrical and hyaline, with measurements ranging from 10.40-15.30 μm. The conidia, both young and
15
16 mature, were observed to have a thick wall. The young conidia were ellipsoid in shape and single-septate, with
17
measurements ranging from 3-6 μm. On the other hand, mature conidia were oval-shaped and exhibited a single
18
19 septum. Their measurements ranged from 10-15 μm in length and 3-7 μm in width. All four isolates (PgDbK02,
20
PgDbK03, PgDbK04 and PgDbK05) exhibited similar cultural and microscopic features (Fig 2).
21
22
23 Pathogenicity assay
24
25
26 In the experiment, the inoculated plants displayed the development of small brown or black patches on the stems
27
28 and yellowing of leaves within 10-12 days after inoculation. As the disease progressed, these dark patches
29 expanded and merged, causing extensive damage to the entire plant. The uninoculated plants remained healthy
30
31 throughout the experiment. These symptoms closely resembled those initially observed on diseased plants in the
32 field. The same pathogen was re-isolated from the diseased plants, thus fulfilling the Koch's postulates.
33
34
35 Phylogenetic analysis
36
37
38 The sequences of all the isolates were analysed by performing a BLAST homology search against the GenBank
39 database at NCBI. It revealed a high similarity of 99.79% with the ITS sequences (MK530072.1) and 99.78%
40
41 with the tef1-α sequences (MN634000.1) of L. theobromae isolates. The sequences obtained in this study,
42 including ITS (OQ874707, OQ874708, OQ874709, and OQ874710) and tef1-α (OQ954065 and OQ954066),
43
44 were submitted to the GenBank database. The concatenated sequence, which combined the ITS and tef1-α
45 regions, comprised a total of 1032 positions in the final dataset. The tef1-α region spanned positions 1 to 504,
46
47 while the ITS region covered positions 505 to 1032, including gaps after alignment. Phylogenetic analysis
48 demonstrated that the isolate (PgDbK03) and L. theobromae are clustered together and formed a distinct lineage
49
50 within the Lasiodiplodia genus with a bootstrap support of 82, as illustrated in the Fig 3. The phylogenetic tree
51 is deposited in TreeBASE under submission ID- 30571, with reviewer access URL-
52
53 http://purl.org/phylo/treebase/phylows/study/TB2:S30571?x-access-
54 code=d0fc87bb44e7b9a4443461d2fcce5482&format=html.
55
56
57 Discussion
58
59
60
61
62 Page 5 of 15
63
64
65
 L. theobromae is recognized as a prominent fungal pathogen due to its capability to infect a wide variety of host
1 plants (Phillips et al. 2013). Its cosmopolitan distribution enables it to exist as latent endophytes within plants
2
3 without causing visible symptoms (Chen et al. 2015). The epidemiological studies conducted on L. theobromae
4 indicate its presence in diverse geographical regions worldwide. However, the extensive research and long-
5
6 standing history of this fungal species has led to inconsistencies in its naming and classification. Over time, it
7 has been assigned different names and treated as distinct species, leading to confusion and ambiguity regarding
8
9 its taxonomic classification and nomenclature. Fortunately, the publication of monograph by Punithalingam
10
(1976) played a crucial role in resolving this confusion. The monograph extensively documented and compiled
11
12 all the synonymous names associated with L. theobromae, providing more accurate identification and
13
understanding of the pathogen's characteristics and behaviour.
14
15
16 In this study, the identification of fungal pathogen responsible for dieback disease in P. graveolens was
17
18 successfully achieved with L. theobromae being identified as the causal agent. The research involved a
19 comprehensive survey conducted over a period of three years, encompassing fifty-two fields located in eight
20
21 different locations. The survey specifically focused on P. graveolens cv. Bourbon and var. CIM Pawan
22 prevalent for cultivation among farmers in India. The survey conducted over multiple years and locations has
23
24 provided valuable information regarding the distribution and occurrence of the disease. The positive results of
25 the pathogenicity assay conducted with all four isolates supported the hypothesis that the observed symptoms
26
27 were specifically caused by the pathogen introduced during the inoculation process. This fulfilment of Koch's
28
postulates provides definitive evidence that the identified pathogen is indeed responsible for the dieback disease
29
30 observed in the geranium plants. By studying the disease incidence and its association with L. theobromae,
31
researchers can gain insights into the factors influencing the disease dynamics and devise appropriate
32
33 management strategies.
34
35
36 The cultural morphology of the fungus observed in this study closely resembles that of L. theobromae. Specific
37 characteristics, such as the presence of aseptate paraphyses and mature conidia with a single septum, distinguish
38
39 it from other species within the Lasiodiplodia genus. It is worth noting that the color, shape, and striations of the
40 conidia also differ from those of other related species. While the size of conidia typically reported for L.
41
42 theobromae falls within the range of 27-35 × 15-20 μm according to previous studies (Table 2) (Pavlic et al.
43 2004; Alves et al. 2008; Coutinho et al. 2017), the dimensions of the conidia in this particular study differ (10-
44
45 15 × 3-7 μm). However, it is important to consider that variations in conidia dimensions have been reported in
46 relation to geographical locations. Maciel et al. (2015) documented variations in the size and shape of L.
47
48 theobromae conidia associated with seeds of Pinus spp. in Brazil. Similarly, Adeniyi et al. (2016) observed
49
differences in conidia size during their investigation of L. theobromae dieback infection on Cashew in Nigeria.
50
51
52 The disease severity observed in this study was significant, ranging from 54% to 78%. Such high levels of
53
54 infection can lead to substantial yield losses, causing considerable hardship for farmers. Similar levels of
55 devastation caused by L. theobromae have been reported by Sulaiman et al. (2012) in severe outbreaks in
56
57 Jatropha curcas plantations, resulting in over 80% losses in Malaysia. More recently, the fungus has been
58 identified as a causative agent for the deterioration of mango and other tropical fruits (Yang et al. 2021). The
59
60 impact of L. theobromae is not limited to specific crops, as it has been associated with massive agricultural
61
62 Page 6 of 15
63
64
65
 losses and field health deterioration across multiple crops and post-harvest produce (Felix et al. 2016). These
1 findings highlight the significant economic and agricultural implications of L. theobromae infections,
2
3 emphasizing the need for effective management strategies to minimize the losses caused by this pathogen.
4
5
6 Phylogenetic inference based on multiple gene sequences has proven to be a powerful tool in elucidating the
7 taxonomy and species boundaries within the genus Lasiodiplodia (Alves et al. 2008; Wang et al. 2021). In this
8
9 study, the application of phylogenetic analysis has successfully identified that the pathogen isolate has 100%
10 similarity index with the L. theobromae. The use of combined ITS and tef1-α sequence data has allowed for a
11
12 more robust classification of L. theobromae within the species complex. The results of the phylogenetic analysis
13 places the isolate obtained in this study in the same clade as the reference strains of L. theobromae, with a
14
15 bootstrap support value of 82 (Hillis & Bull 1993) providing strong evidence to confirm the identity of the
16 pathogen. This phylogenetic analysis not only confirms the species identification but also helps to elucidate the
17
18 relationships among different Lasiodiplodia species. The support from phylogenetic classification data
19 strengthens the identification and characterization of the pathogen, providing a solid foundation for further
20
21 studies on its biology, ecology, and management. The findings of this study contribute to our knowledge of the
22 etiology and epidemiology of dieback disease in P. graveolens and pave the way for future research and control
23
24 measures.
25
26
27 The control of L. theobromae has conventionally relied on the application of chemical fungicides. However,
28 recent studies, such as the one conducted by Yang et al. (2021), have revealed the emergence of resistance in L.
29
30 theobromae isolates against quinone-outside inhibitor (QoI) and methyl benzimidazole carbamates (MBC)
31 fungicides. This resistance is believed to be a consequence of the excessive use of these fungicides in the
32
33 management of stem end rot of mango, highlighting the need for alternative control strategies (Li Y et al. 2020;
34 He et al. 2021). The development of resistance in fungal pathogens poses a significant challenge for disease
35
36 management and emphasizes the urgency to explore alternative approaches, such as bio-control, using native
37 organisms. By harnessing the natural biological interactions, bio-control offers a sustainable and
38
39 environmentally friendly option for disease management (Kamil et al. 2018; Salvatore et al. 2020; Thambugala
40 et al. 2020). Further research and development in bio-control methods targeting L. theobromae could provide
41
42 effective alternatives to chemical fungicides, reduce the risk of resistance development, and promote the long-
43 term sustainability of disease management strategies.
44
45
46 The findings of this study contribute to the understanding of the host range and pathogenicity of L. theobromae,
47
48 specifically in relation to P. graveolens. It highlights the importance of continued research and surveillance to
49 identify and monitor emerging plant diseases and their causative agents. To the best of our knowledge, this
50
51 study represents the first account of L. theobromae causing dieback disease on P. graveolens.
52
53
54 Conflict of interest
55
56 The authors declare that they have no known conflict of interest that could influence the work reported in this
57
58 paper.
59
60
61
62 Page 7 of 15
63
64
65
 Data availability
1
2
3 Microbial strains and specimens used in this study are available with the corresponding author.
4
5
6 Acknowledgement
7
8 The authors are thankful to the director CSIR-CIMAP, Lucknow for providing lab facilities and constructive
9
10 suggestions during the study. This research work was funded under CSIR-Aroma Mission (HCP-0007) program.
11 The institutional approval number for the manuscript is CIMAP/PUB/2023/33.
12
13
14 Reference
15
16
17 Abdollahzadeh J, Javadi A, Goltapeh EM, Zare R, Phillips AJ (2010) Phylogeny and morphology of four new
18
19 species of Lasiodiplodia from Iran. Pers.: Mol. Phylogeny Evol. Fungi. 25(1),1–0.
20
21 Adeniyi DO, Olufolaji DB, Joseph A (2016) Characteristic Variations in Lasiodiplodia theobromae; Pathogen
22
23 of inflorescence Dieback of Cashew in Growing Ecologies of Nigeria. Annu. Res. Rev. Biol. 20,1–6.
24
25
26 Alves A, Crous PW, Correia A, Phillips AJL (2008) Morphological and molecular data reveal cryptic species in
27 Lasiodiplodia theobromae. Fungal Divers. 28, 1– 13.
28
29
30 Androutsopoulou C, Christopoulou SD, Hahalis P, Kotsalou C, Lamari FN, Vantarakis A. (2021) Evaluation of
31
essential oils and extracts of rose geranium and rose petals as natural preservatives in terms of toxicity,
32
33 antimicrobial, and antiviral activity. Pathog. 10(4):494.
34
35
36 Begoude BA, Slippers B, Wingfield MJ, Roux J (2010) Botryosphaeriaceae associated with Terminalia catappa
37 in Cameroon, South Africa and Madagascar. Mycol. Prog. 9(1),101–23.
38
39
40 Ben Hsouna A, Hamdi N (2012) Phytochemical composition and antimicrobial activities of the essential oils
41
and organic extracts from Pelargonium graveolens growing in Tunisia. Lipids Health Dis. 11–167–167.
42
43
44 Blerot B, Baudino S, Prunier C, Demarne F, Toulemonde B, Caissard JC (2016) Botany, agronomy and
45
46 biotechnology of Pelargonium used for essential oil production. Phytochem Rev. 15,935–60.
47
48
49 Boukhris M, Bouaziz M, Feki I, Jemai H, El Feki A, Sayadi S (2012) Hypoglycemic and antioxidant effects of
50 leaf essential oil of Pelargonium graveolens L'Hér. in alloxan induced diabetic rats. Lipids Health Dis.
51
52 11,81–81.
53
54 Burgess TI, Barber PA, Mohali S, Pegg G, Beer W de, Wingfield MJ (2006) Three new Lasiodiplodia spp. from
55
56 the tropics, recognized based on DNA sequence comparisons and morphology. Mycol. 98, 423–435.
57
58
59
60
61
62 Page 8 of 15
63
64
65
 Chen S, Liu Z, Li H, Xia G, Lu Y, He L, Huang S, She Z (2015) β-Resorcylic acid derivatives with α-
1 glucosidase inhibitory activity from Lasiodiplodia sp. ZJ-HQ1, an endophytic fungus in the medicinal
2
3 plant Acanthus ilicifolius. Phytochem. Lett.13,141–6.
4
5
6 Chen Y, Wei H, Du G, Zhu L, Song Q, Hu Y, Wang E, Wang M, Fan X (2018) First report of Lasiodiplodia
7 theobromae causing stem canker on common fig (Ficus carica) in Zhejiang Province of China. Plant Dis.
8
9 102:2656-7.
10
11 Coutinho IB, Freire FC, Lima CS, Lima JS, Gonçalves FJ, Machado AR, Silva AM, Cardoso JE (2017)
12
13 Diversity of genus Lasiodiplodia associated with perennial tropical fruit plants in northeastern Brazil.
14
Plant Pathol. 66(1),90–104.
15
16
17 Damm U, Crous PW, Fourie PH (2007) Botryosphaeriaceae as potential pathogens of Prunus in South Africa,
18
19 with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov. Mycol. 99: 664–680.
20
21
de Silva NI, Phillips AJ, Liu JK, Lumyong S, Hyde KD (2019) Phylogeny and morphology of Lasiodiplodia
22
23 species associated with Magnolia forest plants. Sci. Rep. 9(1):1-1.
24
25
26 Dissanayake AJ, Phillips AJ, Li XH, Hyde KD (2016) Botryosphaeriaceae: Current status of genera and species.
27 Mycosphere. 7(7):1001-73.
28
29
30 Dissanayake AJ, Zhang W, Mei L, Chukeatirote E, Yan JY, Li X, Hyde KD (2015) Lasiodiplodia
31
32 pseudotheobromae causes pedicel and peduncle discolouration of grapes in China. Australas. Plant Dis.
33 Notes. 10:1-5.
34
35
36 Dou ZP, He W, Zhang Y (2017) Does morphology matter in taxonomy of Lasiodiplodia? An answer from
37 Lasiodiplodia hyalina sp nov. Mycosphere. 8(2):1014-27.
38
39
40 Douanla-Meli C, Scharnhorst A (2021) Palm Foliage as Pathways of Pathogenic Botryosphaeriaceae Fungi and
41
42 Host of New Lasiodiplodia Species from Mexico. Pathog. 10(10):1297.
43
44 El-Ganainy SM, Ismail AM, Iqbal Z, Elshewy ES, Alhudaib KA, Almaghasla MI, Magistà D (2022) Diversity
45
46 among Lasiodiplodia Species Causing Dieback, Root Rot and Leaf Spot on Fruit Trees in Egypt, and a
47
Description of Lasiodiplodia newvalleyensis sp. nov. J. Fungi. 8(11):1203.
48
49
50 Felix C, Duarte AS, Vitorino R, Guerreiro AC, Domingues P, Correia AC, Alves A, Esteves AC (2016)
51
52 Temperature modulates the secretome of the phytopathogenic fungus Lasiodiplodia theobromae. Front.
53 Plant Sci. 7:1096.
54
55
56 Felix C, Salvatore MM, DellaGreca M, Ferreira V, Duarte AS, Salvatore F, Naviglio D, Gallo M, Alves A,
57
58 Esteves AC, Andolfi A (2019) Secondary metabolites produced by grapevine strains of Lasiodiplodia
59 theobromae grown at two different temperatures. Mycol. 111(3):466-76.
60
61
62 Page 9 of 15
63
64
65
 Guajardo J, Riquelme N, Tapia L, Larach A, Torres C, Camps R, Besoain X (2018) First report of Lasiodiplodia
1 theobromae causing bot gummosis in Citrus limon in Chile. Plant Dis. 102(4):818.
2
3
4 Harborne JB, Williams CA (2002) Phytochemistry of the genus Geranium. In Geranium and Pelargonium. 32-
5
6 41. CRC Press.
7
8 He R, Yang Y, Hu Z, Xue R, Hu Y (2021) Resistance mechanisms and fitness of pyraclostrobin-resistant
9
10 isolates of Lasiodiplodia theobromae from mango orchards. Plos one. 16(6):e0253659.
11
12
13 Hillis DM, Bull JJ (1993) An empirical test of bootstrapping as a method for assessing confidence in
14 phylogenetic analysis. Syst. Biol. 42(2):182-92.
15
16
17 Hurrah IA, Wagh VV (2021) Revisiting the taxonomy of Geranium kishtvariense (Geraniaceae) & notes on
18
neotypification. Phytotaxa. 516(2):169-77.
19
20
21 Ismail AM, Cirvilleri G, Polizzi G, Crous PW, Groenewald JZ, Lombard L (2012) Lasiodiplodia species
22
23 associated with dieback disease of mango (Mangifera indica) in Egypt. Australas. Plant Pathol..
24 41(6):649-60.
25
26
27 Jayasiri SC, Hyde KD, Jones EB, McKenzie EH, Jeewon R, Phillips AJ, Bhat DJ, Wanasinghe DN, Liu JK, Lu
28
YZ, Kang JC (2019) Diversity, morphology and molecular phylogeny of Dothideomycetes on decaying
29
30 wild seed pods and fruits. Mycosphere. 10:1-86.
31
32
33 Jiang N, Wang XW, Liang YM, Tian CM (2018) Lasiodiplodia cinnamomi sp. nov. from Cinnamomum
34 camphora in China. Mycotaxon. 133(2):249-59.
35
36
37 Kamil FH, Saeed EE, El-Tarabily KA, Abu Qamar SF (2018) Biological control of mango dieback disease
38
39 caused by Lasiodiplodia theobromae using streptomycete and non-streptomycete actinobacteria in the
40 United Arab Emirates. Front. Microbiol. 9:829.
41
42
43 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis
44 across computing platforms. Mol. Biol. Evol. 35(6):1547.
45
46
47 Li Y, Tsuji SS, Hu M, Câmara MP, Michereff SJ, Schnabel G, Chen F (2020) Characterization of
48
49 difenoconazole resistance in Lasiodiplodia theobromae from papaya in Brazil. Pest Manag. Sci..
50 76(4):1344-52.
51
52
53 Linaldeddu BT, Deidda A, Scanu B, Franceschini A, Serra S, Berraf-Tebbal A, Zouaoui Boutiti M, Ben Jamâa
54
ML, Phillips AJ (2015) Diversity of Botryosphaeriaceae species associated with grapevine and other
55
56 woody hosts in Italy, Algeria and Tunisia, with descriptions of Lasiodiplodia exigua and Lasiodiplodia
57
58 mediterranea sp. nov. Fungal Divers. 71(1):201-14.
59
60
61
62 Page 10 of 15
63
64
65
 Liu JK, Phookamsak R, Doilom M, Wikee S, Li YM, Ariyawansha H, Boonmee S, Chomnunti P, Dai DQ, Bhat
1 JD, Romero AI (2012) Towards a natural classification of Botryosphaeriales. Fungal Divers. 57:149-210.
2
3
4 Machado AR, Pinho DB, Pereira OL (2014) Phylogeny, identification and pathogenicity of the
5
6 Botryosphaeriaceae associated with collar and root rot of the biofuel plant Jatropha curcas in Brasil,
7 with a description of new species of Lasiodiplodia. Fungal Divers. 67, 231– 47.
8
9
10 Maciel CG, Muniz MF, Mezzomo R, Reiniger LR (2015) Lasiodiplodia theobromae associated with seeds of
11 Pinus spp. originated from the northwest of Rio Grande do Sul, Brazil. Sci. For. 43(107):639-646.
12
13
14 Meng CR, Zhang Q, Yang ZF, Geng K, Zeng XY, Chethana KT, Wang Y (2021) Lasiodiplodia syzygii sp. nov.
15
16 (Botryosphaeriaceae) causing post-harvest water-soaked brown lesions on Syzygium samarangense in
17 Chiang Rai, Thailand. Biodivers. Data J. 9.
18
19
20 Munera JDC, Hausbeck MK (2016) Characterization of Pythium species associated with greenhouse floriculture
21
crops in Michigan. Plant Dis. 100(3):569-76.
22
23
24 Narnoliya LK, Jadaun JS, Singh SP (2019) The Phytochemical Composition, Biological Effects and
25
26 Biotechnological Approaches to the Production of High-Value Essential Oil from Geranium. Essential
27 Oil Research: Trends in Biosynthesis, Analytics, Industrial Applications and Biotechnological
28
29 Production.327-352.
30
31
32 Nayak AK, Babu BK (2019) First report of powdery mildew caused by Podosphaera fusca on Euphorbia hirta
33 in Odisha state, India. J of Plant Pathol. 101(1):191.
34
35
36 Netto MS, Assunção IP, Lima GS, Marques MW, Lima WG, Monteiro JH, de Queiroz Balbino V, Michereff SJ,
37 Phillips AJ, Câmara MP (2014) Species of Lasiodiplodia associated with papaya stem-end rot in Brazil.
38
39 Fungal Divers. 67(1):127-41.
40
41
42 Nishad I, Srivastava AK, Saroj A, Babu BK, Samad A (2018) First report of root rot of Nepeta cataria caused
43 by Macrophomina phaseolina in India. Plant dis. 102(11):2380.
44
45
46 Osorio JA, Crous CJ, De Beer ZW, Wingfield MJ, Roux J (2016) Endophytic Botryosphaeriaceae, including
47
five new species, associated with mangrove trees in South Africa. Fungal Biol..
48
49
50 Pandey P, Upadhyay RK, Singh VR, Padalia RC, Kumar R, Venkatesha KT, Tiwari AK, Singh S, Tewari SK
51
52 (2020) Pelargonium graveolens L.(Rose-scented geranium): New hope for doubling Indian farmers’
53 income. Environ. Conserv. J.. 21(1&2):141-6.
54
55
56 Pavlic D, Slippers B, Coutinho TA, Gryzenhout M, Wingfield MJ (2004) Lasiodiplodia gonubiensis sp. nov., a
57
58 new Botryosphaeria anamorph from native Syzygium cordatum in South Africa. Stud. Mycol.50: 313–
59 322.
60
61
62 Page 11 of 15
63
64
65
 Pavlic D, Wingfield MJ, Barber P, Slippers B, Hardy GE, Burgess TI (2008) Seven new species of the
1 Botryosphaeriaceae from baobab and other native trees in Western Australia. Mycologia. 100(6):851-66.
2
3
4 Phillips AJ, Alves A, Abdollahzadeh J, Slippers B, Wingfield MJ, Groenewald JZ, Crous PW (2013) The
5
6 Botryosphaeriaceae: genera and species known from culture. Stud. Mycol.. 76(1):51-167.
7
8 Prasad D, Singh A, Singh KP, Bist S, Tewari A, Singh UP (2010) The role of phenolic compounds in disease
9
10 resistance in geranium. Arch. Phytopathol. Pflanzenschutz 43(7):615-23.
11
12
13 Punithalingam E (1976) Botryodiplodia theobromae. [Descriptions of Fungi and Bacteria]. Descriptions of
14 Fungi and Bacteria. 52:Sheet-519.
15
16
17 Punithalingam E (1980) Plant diseases attributed to Botryodiplodia theobromae Pat. J. Cramer.
18
19
20 Rajeswara Rao BR, Bhattacharya AK, Singh HB, Mallavarapu GR (1999) The impact of wilt disease on oil
21 yield and quality of two cultivars of rose-scented geranium (Pelargonium species). J. Essent. Oil Res..
22
23 11: 769-775.
24
25
Rajeswara Rao BR, Kaul PN, Mallavarapu GR, Ramesh S (2000a) First observation of little leaf disease and its
26
27 impact on the yield and composition of the essential oil of rose‐ scented geranium (Pelargonium sp.).
28
Flavour Fragr J. 15(3):137-40.
29
30
31 Rajeswara Rao BR, Sastry KP, Saleem SM, Prakasa Rao EV, Syamasundar KV, Ramesh S. (2000b) Volatile
32
33 flower oils of three genotypes of rose‐ scented geranium (Pelargonium sp.). Flavour Fragr J. 15(2):105-
34 7.
35
36
37 Rajeswara Rao BR (2009) Chemical composition and uses of Indian rose-scented Geranium (Pelargonium
38
39 species) essential oil-A review. J. Essent. Oil-Bear. Plants. 12(4):381-94.
40
41 Rodriguez-Gálvez E, Guerrero P, Barradas C, Crous PW, Alves A (2017) Phylogeny and pathogenicity of
42
43 Lasiodiplodia species associated with dieback of mango in Peru. Fungal Biol.. 121(4):452-65.
44
45
46 Salvatore MM, Alves A, Andolfi A (2020) Secondary metabolites of Lasiodiplodia theobromae: Distribution,
47 chemical diversity, bioactivity, and implications of their occurrence. Toxins. 12(7):457.
48
49
50 Shawl AS, Kumar T, Chishti N, Shabir S (2006) Cultivation of rose-scented geranium (Pelargonium sp) as a
51
cash crop in Kashmir valley. Asian J. Plant Sci.. 5(4):673-5.
52
53
54 Slippers B, Roux J, Wingfield MJ, Van der Walt FJ, Jami F, Mehl JW, Marais GJ (2014) Confronting the
55
56 constraints of morphological taxonomy in the Botryosphaeriales. Pers.: Mol. Phylogeny Evol. Fungi.
57 33(1):155-68.
58
59
60
61
62 Page 12 of 15
63
64
65
 Sulaiman R, Thanarajoo SS, Kadir J, Vadamalai G (2012) First report of Lasiodiplodia theobromae causing
1 stem canker of Jatropha curcas in Malaysia. Plant Dis. 96(5):767.
2
3
4 Thambugala KM, Daranagama DA, Phillips AJ, Kannangara SD, Promputtha I (2020) Fungi vs. fungi in
5
6 biocontrol: An overview of fungal antagonists applied against fungal plant pathogens. Front. Cell. Infect.
7 Microbiol.. 10:604923.
8
9
10 Trakunyingcharoen T, Lombard L, Groenewald JZ, Cheewangkoon R, To-Anun C, Crous PW (2015)
11 Caulicolous Botryosphaeriales from Thailand. Pers.: Mol. Phylogeny Evol. Fungi. 34(1):87-99.
12
13
14 Urbez-Torres JR, Peduto F, Striegler RK, Urrea-Romero KE, Rupe JC, Cartwright RD, Gubler WD (2012)
15
16 Characterization of fungal pathogens associated with grapevine trunk diseases in Arkansas and Missouri.
17 Fungal Divers. 52(1):169-89.
18
19
20 Vardhana J, Kathiravan G, Dhivya R (2017) Biodiversity of endophytic fungi and its seasonal recurrence from
21
some plants. Res J of Phar and Tech. 10(2):490-6.
22
23
24 Wagh VV, Hurrah IA (2020) A new species of Geranium (Geraniaceae) from Himachal Pradesh, India.
25
26 Phytotaxa. 452(1):75-82.
27
28
Wang Y, Lin S, Zhao L, Sun X, He W, Zhang Y, Dai YC (2019) Lasiodiplodia spp. associated with Aquilaria
29
30 crassna in Laos. Mycol. Prog.. 18:683-701.
31
32
33 Wang Y, Zhang Y, Bhoyroo V, Rampadarath S, Jeewon R (2021) Multigene phylogenetics and morphology
34 reveal five novel Lasiodiplodia species associated with blueberries. Life. 11(7):657.
35
36
37 Wu N, Dissanayake AJ, Chethana KT, Hyde KD, Liu JK (2021) Morpho-phylogenetic evidence reveals
38
39 Lasiodiplodia chiangraiensis sp. nov. (Botryosphaeriaceae) associated with woody hosts in northern
40 Thailand. Phytotaxa. 508(2):142-54.
41
42
43 Xiao XE, Wang W, Crous PW, Wang HK, Jiao C, Huang F, Pu ZX, Zhu ZR, Li HY (2021) Species of
44 Botryosphaeriaceae associated with citrus branch diseases in China. Pers.: Mol. Phylogeny Evol. Fungi.
45
46 47(1):106-35.
47
48
49 Yang T, Groenewald JZ, Cheewangkoon R, Jami F, Abdollahzadeh J, Lombard L, Crous PW (2017) Families,
50 genera, and species of Botryosphaeriales. Fungal Biol. 121(4):322-46.
51
52
53 Yang Y, Dong G, Wang M, Xian X, Wang J, Liang X (2021) Multifungicide resistance profiles and biocontrol
54
in Lasiodiplodia theobromae from mango fields. Crop Prot. 145:105611.
55
56
57 Zhang W, Groenewald JZ, Lombard L, Schumacher RK, Phillips AJ, Crous PW (2021) Evaluating species in
58
59 Botryosphaeriales. Pers.: Mol. Phylogeny Evol. 46(1):63-115.
60
61
62 Page 13 of 15
63
64
65
 Zhao L, Wang Y, He W, Zhang Y (2019) Stem Blight of Blueberry Caused by Lasiodiplodia vaccinii sp. nov. in
1 China. Plant Dis. 103(8):2041-50.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 Page 14 of 15
63
64
65
 Legends of Figures and Tables
1
2 Figures
3
4 Fig 1 Geranium plants affected by dieback disease: a-d showing symptoms of natural infection in the field
5
6 during monsoon season of 2017, 2018, and 2019
7
8 Fig 2 Microscopic observations of Lasiodiplodia theobromae (PgDbK03) isolated from Pelargonium graveolens
9 a. fungal hyphae; b. conidiomata and paraphyses arising from an infected branch of geranium; c. d. and e.
10
11 mature conidia; f. asci; g. young conidia h. conidia cells visible in transverse section of infected plant tissue
12
13 Fig 3 Phylogenetic relationship between Lasiodiplodia species, inferred from ITS and tef1-α nucleotide
14 sequence data. Phylogenetic tree constructed using the ML method and HKY model. The robustness of the most
15
16 parsimonious trees was evaluated from 1000 bootstrap replications. The significant Bootstrap values >70 are
17 given at the nodes. This analysis involved 64 nucleotide sequences with a total of 1032 positions in the final
18
19 dataset. The tree is rooted to Diplodia mutila (3-1-51A) and Diplodia corticola (CBS 112073)
20
21
22
23 Tables
24
25 Table 1 Dieback disease incidence in rose scented geranium in open field condition during monsoon seasons
26
27 Table 2 Different Lasiodiplodia species information including morphological observations and sequence data
28
used in this study
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62 Page 15 of 15
63
64
65
Table Click here to access/download;Table;Artwork-PgDbK- J. of
Plant Pathology- Tables.doc

Tables

Table 1

Fields surveyed Disease incidence (%)

Cultivar/
S. No No. of Year
Variety Location
fields 2017 2018 2019
Badaun 4 70 65 75
P. graveolens Sambal 7 68 71 62
1.
cv. Bourbon Rampur 5 78 67 59
Barabanki 10 55 64 60
Sitapur 4 71 66 70
P. graveolens Shahjahanpur 7 70 68 65
2.
var. CIM Pawan Sambal 5 64 73 59
Badaun 10 60 54 56

Page 1 of 5
Table 2
Sequences used in this study
Species Host Isolate name Conidia size Reference
ITS tef1-α

(21.5-)25-29.5(-31) × Zhang et al.

L. acaciae Acacia spp. CBS:136434 MT587421.1 MT592133.1
(11)12-14(-15) μm 2021

(23–)25–28(–29) × Wang et al.

L. aquilariae Aquilaria crassna GuoLD01961 KY783442.1 KY848600.1
12–16 μm 2019

(20-) 25-26 (-29) × Osorio et al.

L. avicenniae Avicennia marina LAS199 KU587957.1 KU587947.1
(9-) 11-11.5 (-15) μm 2016

MFLUCC 17- Jayasiri et al.

L. avicenniarum A. marina NR_163344.1 MK340867.1 26–32 × 11–14 μm
2591 2019

22.7–29.2 × 11.7– Netto et al.

L. brasiliensis Citrullus lanatus CFC-1123 OL890691.1 OL841380.1
17.0 µm 2014

Bruguiera (19-) 25-26 (-32) × Osorio et al.

L. bruguierae CMW41614 KP860834.1 KP860679.1
gymnorrhiza (11-) 12-13 (-15) µm 2016

Eriobotrya 13–20.2 × 10.1–12.6 Coutinho et

L. caatinguensis GB74 MT981854.1 MW315804.1
japonica µm al. 2017

Saprobic on GZAAS 21-00031, 1 2

Wu et al.
L. chiangraiensis MW760853.1 MW815629.1 NA
woody hosts GZAAS 21-00142 2021

Vaccinium Dou et al.

L. chinensis CGMCC3.18061 KX499889.1 KX499927.1 19–25 × 12–14 µm
uliginosum 2017

(17.5–) 18.7-21.1 (–
Cinnamomum Jiang et al.
L. cinnamomi CFCC 51997 MG866028.1 MH236799.2 22.4) × (11.5–) 12.7-
camphora 2018
14.1 (–15.5) μm

22.5 – 26.6 × 13.6 – Abdollahzade

L. citricola Citrus spp. XGWY42 MT849762.1 MT856964.1
17.2 μm h et al. 2010

(28–) 29–36 (–38) × Wang et al.

L. clavispora V. uliginosum CGMCC3.19594 MK802166.1 OL773697.1
12–15μm 2021

Burgess et al.
L. crassispora Santalum album GB5 MT981840.1 MW315790.1 27– 30 × 14 –17 μm
2006

(18–) 20–24 (–25) × Wang et al.

L. curvata A. crassna GuoLD01755 KY783443.1 KY848601.1
12–15μm 2019

Ismail et al.
L. egyptiacae Mangifera indica CMM3648 KF234549.1 KF226705.1 22 × 12 μm
2012

MFLUCC 18- de Silva et al.

L. endophytica Magnolia spp. MK501838.1 MK584572.1 NA
1121 2019

Page 2 of 5
 Machado et
L. euphorbicola Jatropha curcas GB180 MT981878.1 MW315827.1 15–23 × 9–12 μm
al. 2014

(19.6–)21.8(−24.3) ×
Linaldeddu et
L. exigua Retama raetam CMW36131 KU887267.1 KU886881.1 (10.8–) 12.3(−13.3)
al. 2015
μm

(22–) 23–29 (–30) × Wang et al.

L. fujianensis V. uliginosum CGMCC:3.19593 MK802164.1 MK887178.1
(12–) 13–15 (–16) μm 2021

28.6–33.4 × 15.6– Abdollahzade

L. gilanensis On woody twigs UCD9144 MZ246641.1 OM387009.1
17.6 μm h et al. 2010

Syzygium Pavlic et al.

L. gonubiensis MBA32 KY052916.1 KY024601.1 32–36 × 16–18.5 μm
cordatum 2004

(23–)28–31(–33.5) ×
BE591, 1 2
Xiao et al.
L. guilinensis Citrus spp. 2
MW880673.1 MW884175.1 (13.5–) 15–16.5(–17)
BE31 2021
μm

19.6 – 23.4 × 11.7– Abdollahzade

L. hormozganensis M. indica CDA1363 KY994628.1 KX528571.1
13.3 μm h et al. 2010

Salvadora 18.7– 22.7 × 12.1– Abdollahzade

L. iranensis CDA1147 KY994620.1 KX528563.1
persica, 13.9 μm h et al. (2010)

(20–) 22–29 (–30) × Wang et al.

L. irregularis A. crassna GuoLD01673 KY783472.1 KY848610.1
(12–) 13 (–15) μm 2019

Machado et
L. jatrophicola J. curcas CDA1146 KY994634.1 KX528560.1 22−26 × 14−17 μm
al. 2014

(18-)22.8(-27.4) × Rodriguez-
L. laeliocattleyae M. indica ARM29 MK480475.1 MK495373.1 (11.7-) 14.6(-17.2) Gálvez et al.
μm 2017

(23–) 24–28 (–30) × Wang et al.

L. laosensis A. crassna GuoLD01818 KY783471.1 KY848609.1
(13–) 14–15 (–17) μm 2019

VMT281, Wang et al.

L. lignicola A. crassna 2
MW940855.11 KU887003.12 15–17.5 × 8– 11 μm
CBS 134112 2019

(24.5–)27–30(–32) ×
Xiao et al.
L. linhaiensis Citrus spp. BE52 MW880683.1 MW884186.1 (12.5–) 13.5–15(–16)
2021
μm

Lodoicea 16.7–19.5 × 8.4–9.5 Douanla-Meli

L. lodoiceae AGQMy0006 MW274146.1 MW604229.1
maldivica μm et al. 2021

(26–) 28–34 (–36) × Wang et al.

L. macroconidia A. crassna GuoLD01752 KY783438.1 KY848597.1
13–16 μm 2019

Machado et
L. macrospora J. curcas CMM3833 KF234557.1 KF226718.1 28−35 × 15−17 μm
al. 2014

Page 3 of 5
 MFLUCC 18- (24-) 25-27(-30) × 11- de Silva et al.
L. magnoliae Magnolia spp. MK499387.1 MK568537.1
0948 15 μm 2019

Terminalia (13.5-)15.5-19(-21.5) Begoude et

L. mahajangana CMW27818 FJ900596.1 FJ900642.1
catappa × (10-) -11.5-13(-14) al. 2010

Adansonia (12-)14-17(-19) × Pavlic et al.

L. margaritacea CBS122519 EU144050.2 EU144066.1
gibbosa. (10-)11-12 (-12.5) μm 2008

19.1– 28.5 × 10–15.3 Netto et al.

L. marypalmiae Carica papaya CMM2274 KC484841.1 KC481567.1
μm 2014

(26.3–)30.6(−37) × Linaldeddu et
L. mediterranea Vitis vinifera 13-1213 KU578250.1 KU695584.1
(13.5–) 16.1(−18) μm al 2015

Chamaedorea Douanla-Meli
L. mexicanensis AGQMy0015 MW274150.1 MW604233.1 NA
seifrizii et al. 2021

(18–) 19–22 (–23) × Wang et al.

L. microconidia A. crassna BE80 MW880670.1 MW884173.1
10–15 μm 2019

(16.1-)17.4-19.6(-21)
Urbez-Torres
L. missouriana V. vinifera UCD2193MO HQ288225.1 HQ288267.1 × (8.1-) 8.9- 10.6(-
et al. 2012
11.8) μm
(20–) 21–26 (–28) ×
Wang et al.
L. nanpingensis V. uliginosum CGMCC:3.19596 MK802167.1 OL773698.1 13–16
2021
(–17) μm

18.3 – 22.1 × 10.7– Alves et al.

L. parva Manihot esculenta UCR1056 JQ659278.1 JQ659265.1
12.3 μm 2008

26.7– 32.5 × 14.4 – Damm et al.

L. plurivora Prunus salicina ID0055 MT649615.1 MT666043.1
16.7 μm 2007

(16–)23.5–27.5(–
Xiao et al.
L. ponkanicola Citrus spp. BE44 MW880685.1 MW884188.1 28.5) × (11–) 13–
2021
14.5(–15.5) μm

Eriobotrya 16.3–26.4 × 9.6–15 Coutinho et

L. pontae CGMCC_3.18051 MK510560.1 MK510671.1
japonica μm al. 2017

L. Alves et al.
Gmelina arborea GXJG4.5 MH487656.1 MH487655.1 26–31 × 13–16 μm
pseudotheobromae 2008

(19–)21.5–25(–28) ×
Slippers et al.
L. pyriformis Acacia mellifera CBS 121770 EU101307.1 EU101352.1 (13.5–) 15.5–19.5(–
2014
21.5) μm

Eucalyptus Burgess et al.

L. rubropurpurea RB07 KY052966.1 KY024637.1 24–33 × 13–17 μm
grandis 2006

(12-)14-16 (-17) × (8- Yang et al.

L. sterculiae Sterculia oblonga CBS:342.78 KX464140.1 KX464634.1
)10-11 (-12) μm 2017

Page 4 of 5
 Machado et
L. subglobosa J. curcas CDA1137 KY994622.1 KX528565.1 16−23 × 11−17 μm
al. 2014

MFLUCC 18- Jayasiri et al.

L. swieteniae Swietenia spp. MK347789.2 MK340870.1 24–32 × 11–14 μm
0244 2019

Syzygium (27-)30-32(-36) × Meng et al.

L. syzygii CBS:120512 MT587434.1 MT592147.1
samarangense (13-)15-17 (-20) μm 2021

(18-)19-24(-26) × Wang et al.

L. tenuiconidia A. crassna GuoLD01857 KY783466.1 KY848619.1
(11-)12-16 (-17) μm 2019

Trakunyingch
BJFU
L. thailandica M. indica KY676788.1 KY676797.1 (20–26 × 12–16 μm) aroen et al.
DZP160119-9
2015
M. indica,
Alves et al.
L. theobromae Phoenix FBG2019_090_1 MT302844.1 MT434994 21–31 × 13–15.5 μm
2008
dactylifera

PgDbK02 OQ874707 NA

Pelargonium PgDbK03 OQ874708 OQ954065

L. theobromae 10-15 μm × 3-7 μm This study
graveolens PgDbK04 OQ874709 OQ954066

PgDbK05 OQ874710 NA

(17-)18-24(-25) × Wang et al.

L. tropica A. crassna GuoLD01846 KY783454.1 KY848616.1
(12-)13-14 (-15) μm 2019

(18-) 21-27 (-31) × Zhao et al.

L. vaccinii Vaccinium spp. CGMCC3.19251 MK157134.1 MK157161.1
(11-)12-14(-16) μm 2019

Burgess et al.
L. venezuelensis Acacia mangium CBS:129755 MH865371.1 EU673305.1 26–33 × 12–15 μm
2006

(16.8-)18.2-20.5(-
22.9) × Urbez-Torres
L. viticola V. vinifera. CMM1472 JX464061.1 JX464040.1
(7.9-)8.8-10.1(-10.7) et al. 2012
μm

(25-)26-28 (-32) × Yang et al.

L. vitis V. vinifera CBS:124060 KX464148.1 KX464642.1
(12-)15-16 (-17) μm 2017

*Diplodia mutila 3-1-51A OP006733.1 OP373139.1

Outgroup
*D. corticola CBS 112073 AY268420.1 MT592041.1

NA- Not available, *-used as outgroup in the phylogenetic studies

Page 5 of 5
Figure Click here to access/download;Figure;Artwork-PgDbK- J. of
Plant Pathology- Figures.doc

Figures

Fig 1

b.

a. c. d.

Page 1 of 3
Fig 2

f
.

a. b.

g.

c. d. e. h.

Page 2 of 3
Fig 3

Page 3 of 3