Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria.

Several Botryosphaeriaceae species are known to occur worldwide, causing dieback, canker and fruit rot on various hosts. Surveys conducted in ten commercial citrus orchards in the northern region of Algeria revealed five species of Botryosphaeriaceae belonging to three genera associated with diseased trees. Morphological and cultural characteristics as well as phylogenetic analyses of the internal transcribed spacer (ITS) region and the translation elongation factor 1-alpha (tef1-α) identified Diplodia mutila, Diplodia seriata, Dothiorella viticola, Lasiodiplodia mediterranea and a novel species which is here described as Lasiodiplodia mithidjana sp. nov.. Of these, L. mithidjana (14.1% of the samples) and L. mediterranea (13% of the samples) were the most widespread and abundant species. Pathogenicity tests revealed that L. mediterranea and D. seriata were the most aggressive species on citrus shoots. This study highlights the importance of Botryosphaeriaceae species as agents of canker and dieback of citrus trees in Algeria.

Introduction

Citrus represent one of the main fruit crops in the world. They are widely recognized by their nutritional and health-related properties of both fresh fruit and juice. Being produced in more than 140 countries, citrus production reached more than 13 million Tonnes, in 2018 [1]. Citrus cultivation is one of the major contributors to the Algerian wealth and is part of the traditional agriculture of the country. Many types of citrus are grown in Algeria, including oranges (48 400 ha), clementines (10 817 ha), mandarins (2 347 ha), lemons (4 409 ha) and grapefruits (83 ha) [2]. Despite the high adaptation capacity of citrus trees to different climates [3], a number of unfavourable factors has led to a decrease of the total citrus yield in Algeria. Among these factors, ageing trees, droughts, inappropriate cultural practices and the effects of various pests and pathogens are the most important [3, 4]. Citrus diseases are numerous and diverse, and are caused by phytopathogenic agents belonging to viruses, viroids, phytoplasmas, bacteria, and fungi [3] Some pathogens cause very serious diseases, predisposing to, and inciting dieback, while others are less serious [3-6].

Recently, trunk diseases have become a growing threat in both, old and newly established orchards of citrus, worldwide. Symptoms include leaves that become yellow and fall early, shoots and twigs die, increasing the risk of citrus decay as the damage expands to the trunk [3, 7–9]. To date, among the fungi that impact citrus, Diaporthe species are well known for causing stem-end rot and melanose of fruits, young leaf and shoot gummosis and blight of perennial branches and trunks, in Greece, Italy, Malta, Portugal, Spain, China, Korea, New Zealand, and the USA [9-13].

Fusarium and Neocosmospora have also been reported causing canker and dieback diseases of citrus, in Tunisia, Greece, Italy and Spain [14-16]. The Diatrypaceae are other canker and dieback pathogens impacting citrus orchards [17]. Several Eutypella spp. have been reported from Citrus sp. In southern California desert, three distinct species of Eutypella are found associated with citrus branch canker, namely: Eutypella citricola, E. microtheca and a Eutypella sp. [17-19].

In addition to the above fungal pathogens that compromise citrus crops, several Botryosphaeriaceae species are known to colonize citrus trees. The Botryosphaeriaceae family is recognized as an important and widely distributed plant pathogen, which impacts on a variety of economically important hosts. It comprises at least 24 genera encompassing 222 known species, living as endophytes, saprobes, or plant pathogens [20, 21]. Recent studies carried out in California, Italy and Tunisia have highlighted the Botryosphaeriaceae as the most prevalent fungi that cause cankers, vascular necrosis and dieback of citrus trees [8, 17, 18, 22]. Adesemoye et al. [23] recovered various Botryosphariaceae species from necrotic tissues of citrus branch canker and rootstock, including, Diplodia seriata, D. mutila, Dothiorella viticola, Doth. iberica, Lasiodiplodia parva, Neofusicoccum australe, N. luteum, N. mediterraneum, N. parvum and Neoscytalidium dimidiatum. In Iran, Abdollahzadeh et al. [24], described Lasiodiplodia citricola from citrus trees showing symptoms of branch dieback.

In Algeria, members of the Botryosphaeriaceae have been reported to cause diseases on Vitis vinifera [25-27], Quercus suber [28] and Cupressus macrocarpa [29]. Linaldeddu et al. [30] isolated and described L. mediterranea from a cankered branch of Citrus sinensis trees in northern Algeria. However, the impact of Botryosphaeriaceae species on citrus trees in Algeria has not been studied in detail. Therefore, the aim of this study was to investigate and determine the incidence as well as evaluating the pathogenicity of the Botryosphaeriaceae species associated with branch canker and dieback in the major citrus-growing region of Algeria.

Materials and methods

Ethics statement

No specific permits were required for the described field studies. This study did not involve endangered or protected species.

Field survey and sampling

Surveys were conducted in ten commercial orchards in the northern region of Algeria. Specifically, in the Mitidja plain at the base of the Tell Atlas Mountains. The sampling was done in four municipalities; namely Oued El Aleug (4 orchards), Chiffa (2 orchards), Boufarik (2 orchards) and the coastal town, Sidi Fredj, located within the territory of the Staoueli municipality, situated by the Mediterranean Sea (2 orchards). The field diagnosis and sampling were performed between April 2013 and March 2015. Samples were collected from the orchards with permission of landowners. All the prospected orchards had approximately the same age (between 25 and 30 years old). Trunks and branches showing symptoms such as dead shoots, defoliation, cankers, wood necrosis, and dieback were collected, randomly. A total of 80 symptomatic sweet orange (Citrus sinensis) trees were sampled (Table 1).

Table 1

Citrus orchards surveyed and number of samples collected.

Locality	Orchards	Area (ha)	Number of trees sampled	Number of samples processed
Oued El Alleug	a	18	5	9
	b	16	5	6
	c	28	5	7
	d	6.8	5	6
Chiffa	a	25	10	13
	b	18	10	10
Boufarik	a	43	10	10
	b	27	10	11
Staoueli	a	32	10	10
	b	15	10	10

Fungal isolation and morphological characterization

In the laboratory, all samples were processed by peeling the outer bark surface with a sterilized scalpel. Longitudinal and transversal cuts were made to reveal the type and localization of the internal necrosis. From each lesion detected, ten pieces of wood, approx. 5 mm2, were cut from the margins between necrotic and healthy tissues. These pieces were submerged in 4% sodium hypochlorite for 15 min, washed thrice with sterile distilled water, dried with sterilized filter paper and placed onto the surface of potato dextrose agar (PDA, Difco Laboratories). Plates were incubated at 25°C until growth was detected. The mycelium emerging from wood pieces were transferred onto fresh PDA plates and incubated under the same conditions.

Preliminary identifications to genus and tentative species level were based on colony and conidial morphology (colony colour, colony growth pattern, conidial size, shape, colour, striation, septation, conidiogenous cells, and presence of paraphyses) according to Phillips et al. [20]. Isolates that lacked pycnidia production on PDA were placed on autoclaved pine needles in ¼ strength PDA within 2–3 weeks, incubated at 25°C under mixed near-UV and cool-white fluorescent light in a 12 h light 12 h dark regime for 2–6 weeks, to enhance fruiting body production. Conidiogenous layer and conidia were mounted in 100% lactic acid and observed with a Nikon 80i light microscope.

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from 7 days old axenic cultures, grown on PDA at 25°C, following Santos and Phillips [31]. PCR reactions were carried out with Taq DNA polymerase, nucleotides and buffers supplied by MBI Fermentas (Vilnius, Lithuania). PCR reaction mixtures were prepared as previously described by Alves et al. [32], with the addition of 5% DMSO to improve the amplification of some difficult DNA templates. The ITS region plus D1/D2 domain of the LSU was amplified with the primer pair ITS1 [33] and NL4 [34]. The amplification conditions were initial denaturation of 5 min at 95°C, followed by 29 cycles of 30 s at 94°C, 30 s at 50°C, and 1.5 min at 72°C, and a final extension of 10 min at 72°C. Part of the translation elongation factor 1 alpha gene (tef1-α) was amplified with primers EF1-688F and EF1-1251R [35]. The amplification conditions were: initial denaturation of 5 min at 95 C, followed by 30 cycles of 30 s at 94°C, 45 s at 55°C, 1½ min at 72°C, and a final extension period of 10 min at 72°C. ITS and tef1-α regions were sequenced in both directions by STAB Vida Lda (Portugal), using the Sanger method.

The nucleotide sequences were read and edited with BioEdit Alignment Editor V.7.0.9.0 [36]. Newly generated sequences were deposited in GenBank (Table 2). Homological sequences of the newly sequenced ones were retrieved from the GenBank using the Basic Local Alignment Search Tool (BLAST) [37].

Table 2

Botryosphaeriaceae species included in this study.

Species	Isolate number	Host/ Substrate	Origin	Collector	GenBank accession numbers
					ITS	tef1-α
Lasiodiplodia mediterranea	ALG104	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104094	MN159093
L. mediterranea	ALG105	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104095	MN159094
L. mediterranea	ALG40	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104096	MN159095
L. mediterranea	ALG78	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104097	MN159096
L. mediterranea	ALG41	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104098	MN159097
L. mediterranea	ALG36	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104099	MN159098
L. mediterranea	ALG80	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104100	MN159099
L. mediterranea	ALG106	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104101	MN159100
L. mediterranea	ALG107	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104102	MN159101
L. mediterranea	ALG108	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104103	MN159102
L. mediterranea	CBS 124060	Vitis, wood fragment	Italy, Sicily	S. Burruano	KX464148	MN938928
L. mitidjana	ALG81	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104104	MN159103
L. mitidjana	ALG44	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104105	MN159104
L. mitidjana	ALG39	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104106	MN159105
L. mitidjana	ALG42	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104107	MN159106
L. mitidjana	ALG38	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104108	MN159107
L. mitidjana	ALG43	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104109	MN159108
L. mitidjana	ALG37	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104110	MN159109
L. mitidjana	ALG34	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104111	MN159110
L. mitidjana	ALG82	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104112	MN159111
L. mitidjana	ALG109	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104113	MN159112
L. mitidjana	ALG110	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104114	MN159113
L. mitidjana	ALG111 = MUM 19.90	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104115	MN159114
L. mitidjana	ALG112	Citrus/wood canker	Algeria, Boufarik	Akila Berraf-Tebbal	MN104116	MN159115
Diplodia seriata	ALG93	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104117	MN159116
D. seriata	ALG94	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104118	MN159117
D. seriata	ALG98	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104119	MN159118
D. seriata	ALG91	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104120	MN159119
D. seriata	ALG92	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104121	MN159120
D. seriata	ALG90	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104122	MN159121
D. seriata	ALG89	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104123	MN159122
D. seriata	ALG96	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104124	MN159123
D. seriata	ALG95	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104125	MN159124
D. seriata	ALG97	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104126	MN159125
D. mutila	ALG99	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104127	MN159126
D. mutila	ALG103	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104128	MN159127
D. mutila	ALG100	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104129	MN159128
D. mutila	ALG102	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104130	MN159129
D. mutila	ALG101	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104131	MN159130
Dothiorella viticola	ALG83	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104087	MN159086
Doth. viticola	ALG35	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104088	MN159187
Doth. viticola	ALG84	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104089	MN159188
Doth. viticola	ALG85	Citrus/wood canker	Algeria, Staoueli	Akila Berraf-Tebbal	MN104090	MN159189
Doth. viticola	ALG86	Citrus/wood canker	Algeria, Oued El Alleug	Akila Berraf-Tebbal	MN104091	MN159190
Doth. viticola	ALG87	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104092	MN159191
Doth. viticola	ALG88	Citrus/wood canker	Algeria, Chiffa	Akila Berraf-Tebbal	MN104093	MN159192

Phylogenetic analysis

Sequences of all Lasiodiplodia species known from culture were retrieved from GenBank (S1 Table) and aligned with sequences of the isolates obtained in this study. Alignments were done with ClustalX v. 1.83 [38] using the following parameters: pairwise alignment parameters (gap opening = 10, gap extension = 0.1) and multiple alignment parameters (gap opening = 10, gap extension = 0.2, transition weight = 0.5, delay divergent sequences = 25%). Alignments were checked and manual adjustments made if necessary using BioEdit v. 7.2.5 [36]. Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses were performed using MEGAX [39]. The best fitting DNA evolution model was determined also by MEGAX. A discrete Gamma distribution with five categories was used to model evolutionary rate differences among sites (+G, parameter = 0,3394). ML analysis was performed on a Neighbour-Joining starting tree automatically generated by the software. Nearest-Neighbour-Interchange (NNI) was used as the heuristic method for tree inference. MP analysis was done using the Tree-Bisection-Regrafting (TBR) algorithm with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). The robustness of the trees (ML and MP) was evaluated by 1000 bootstrap replications.

Pathogenicity test

Aggressiveness of the fungi was evaluated by measuring the lengths of the internal lesions. The ability of the isolates to cause cankers was assessed in vivo on 1-year-old detached shoots collected from symptomless Citrus sinensis trees, following Hamrouni et al. [22] and Adesemoye et al. [39]. From each phylogenetically resolved species, two representative isolates were selected. The shoots with 25 mm in diameter were cut into equal length (25 cm long). They were then surface disinfected with 70% ethanol and wounded on an intermediate internode, with a scalpel. From each strain, a 5 mm diameter mycelial plug taken from a 5- day old colony growing on PDA was placed into the wound. Negative controls were inoculated with fresh, non-colonized, PDA plugs. The point of inoculation was covered with wet sterile cotton and sealed with Parafilm® to prevent desiccation. Subsequently, the cuttings were well watered and maintained under favorable conditions. There were 10 replicates per isolate, and the same number of cuttings was used as controls. One month after inoculation, lengths of lesions produced by each strain were measured. In an attempt to recover the inoculated fungi and complete Koch’s postulates, necrotic tissue from the margin of the lesions was taken and placed onto PDA.

Statistical analyses

Internal lesion lengths from the pathogenicity test were analysed. Means where checked for normality using Shapiro-Wilk test with α = 0.05, then differences in lesion lengths caused by fungal isolates belonging to different species were assessed using One-way analysis of variance (ANOVA) with P ≤ 0.05. Significant differences with the confidence interval of 95% were detected by applying Tukey's honestly significant difference (HSD) test. The R v. 3.5.1 statistical software was used to perform the statistical analysis.

Nomenclature

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Results

Disease symptoms

Citrus dieback was detected in all the orchards and regions investigated, with different degrees of intensity. Various external symptoms, including partial or complete dieback of the tree, branch and shoot cankers, abnormal growth of epicormic shoots; defoliation and leaf chlorosis were observed. Moreover, in certain orchards, bark cracking of the trunk and the branches was also noticeable (Fig 1).

Fig 1

Citrus tree with dieback symptoms (a), bark cracking of the trunk and gummosis (b), main internal symptoms of sectioned branches and trunks (c–f).

The analysis of the 80 symptomatic sweet orange (Citrus sinensis) trees sampled to carry out the isolations, revealed the existence of 92 necrotic lesions in the trunks and the branches. They belonged to four types of wood alteration, including: wedge-shaped necrosis (WSN), that was the most prevalent lesion (n = 30) of the total samples collected. The brown central necrosis (BCN) (n = 26) was the second most prevalent lesion, followed by the black spots in the xylem (BS) (n = 24) and yellow soft wood rot (YSW) (n = 12).

Fungal isolation and identification

Isolation carried out from ninety-two samples yielded a total of forty-seven fungal colonies belonging to Botryosphaeriaceae. On the basis of morphological characteristics, it was possible to distinguish three morphological groups according to colour and shape of conidia. Twenty-five isolates with brown sub-globose and striate conidia were grouped as Lasiodiplodia-like fungi. Fifteen isolates with brown oblong to ovoid conidia as Diplodia-like fungi. A further seven isolates with brown, ovoid thick walled and 1-septate conidia were considered as Dothiorella-like fungi. The identification of the isolates was confirmed by analysis of ITS and tef1-α sequences, which distinguished five separate species. The BLAST searches in GenBank showed 99–100% identity with reference sequences of representative isolates including that of the ex-type. The identified species were: D. seriata (10 isolates), D. mutila (5 isolates), Doth. viticola (7 isolates), L. mediterranea (10 isolates) and a Lasiodiplodia sp. (14 isolates) that could not be assigned to any of the currently known species.

Phylogenetic analysis was performed using ITS and tef1-α sequences. Fragments of approximately 500 and 300 bases were determined for ITS and tef1-α regions, respectively. The ML and the MP trees are presented in Fig 2 and S1 Fig, respectively. The combined ITS and tef1-α dataset of Lasiodiplodia consisted of 23 isolates aligned with sequences of 69 isolates retrieved from GenBank, representing a selection of all known Lasiodiplodia and 2 outgroup taxa (D. seriata CBS 112555 and D. mutila CBS 112553). In the ML phylogenetic tree (Fig 2), the isolates obtained in this study grouped in two clades. The first clade comprised 10 isolates, which clustered together, with the ex-type strain of L. mediterranea (CBS 137783) and the ex-type strain of L. vitis (CBS 124060) (S1 Table), forming a single monophyletic group. The second group contained 14 isolates, which formed a distinct clade, with a high bootstrap support (ML/MP = 80/94), was considered to represent a distinct species, which is described here as Lasiodiplodia mitidjana sp. nov. (Fig 2).

Fig 2

Maximum likelihood tree generated from the combined analysis of ITS and tef1-α sequence data.

ML/MP bootstrap values are given at the nodes. Support values less than 50% are omitted or indicated with ‘–’. The tree was rooted to D. mutila and D. seriata.

Taxonomy

A. Alves, A.E. Mahamedi & A. Berraf-Tebbal sp. nov. (Fig 3) [urn:lsid:mycobank.org:names: MB 832823]. Algeria, Mitidja, isolated from a branch canker of Citrus sinensis, June 2015, Akila Berraf-Tebbal, HOLOTYPE AVE-F-7, a dried culture sporulating on pine needles twigs deposited in the Herbarium Universitatis Aveirensis (AVE), culture ex-holotype MUM 19.90 (= ALG111). Other isolates examined are listed in Table 2.

Fig 3

Lasiodiplodia mitidjana.

(a,b). Pycnidia formed on pine needles. (c). Conidiogenous layer with conidia developing on conidiogenous cells. (d). Conidia developing on conidiogenous cells and paraphyses. (e,f,i). Hyaline aseptate conidia. (g,h). Hyaline aseptate brown 1-septate conidia in two focal planes showing the striations on the inner surface of the wall. (j). Aseptate conidia, one becoming brown. (k,l). Brown 1-septate conidia in two focal planes to show the striations in the inner surface of the wall. Scale bars: e = 20 μm, c,d,f–j = 10 μm; k–l = 5 μm.

Etymology. named after Mitidja where the fungus was discovered.

Sexual state. Not seen. Asexual state: Conidiomata stromatic, pycnidial, produced on pine needles on ¼ strength PDA within 2–3 wks, dark brown to black, covered with dense mycelium, superficial or immersed in the host becoming erumpent when mature, mostly uniloculate, solitary, globose, thick-walled. Paraphyses hyaline, cylindrical, thin-walled, initially aseptate, becoming septate when mature, rounded at apex. Conidiogenous cells holoblastic, discrete, hyaline, smooth, thin-walled, cylindrical, sometimes slightly swollen at the base. Conidia subovoid to ellipsoid-ovoid, apex rounded, occasionally tapering to truncate base, widest in middle to upper third, thick-walled, with granular content, initially hyaline and aseptate, remaining so for a long time, becoming dark brown and 1-septate, with longitudinal striations, (22.6–)27.7(−31.9) × (13.5–)16.7(−19.6) μm, 95% confidence limits = 27.3–28 × 16.5–16.9 μm (av. of 125 conidia ± SD = 27.7 ± 1.9 × 16.7 ± 1.1 μm, L/W ratio = 1.7).

Cultural characteristics. Colonies on PDA with moderate to dense aerial mycelium, initially white to smoke-grey, turning greenish grey on the surface and reverse, becoming dark slate blue with age.

Cardinal temperatures for growth. Minimum <10°C, maximum < 40°C and optimum 25–35°C, covering the medium surface (90 mm) before 7 days at 25°C in the dark.

Habitat. Twigs and branches of Citrus sinensis.

Known geographic distribution. Algeria.

Notes. Phylogenetically it is very closely related to L. citricola being distinguished by three bp in the tef1-α locus. Conidia tend to be larger than those of L. citricola, 95% confidence limits = 24.1–24.9 × 15–15.7 μm (av. ± S.D. = 24.5 ± 0.2 × 15.4 ± 1.8 μm) and have a lower L/W ratio = 1.6.

Linaldeddu, Deidda & Berraf-Tebbal sp. nov. (Linaldeddu et al. 2015. Fungal diversity 71:207)

MycoBank. MB 808356

Synonym. Yang & Crous, sp. nov. (Yang et al. 2017. Fungal Biology 121)

MycoBank. MB817635

Notes. Yang et al. [40] described L. vitis as a novel species, clearly distinct from the species recognised on Vitis vinifera in Italy. However, in their study Yang et al. [40] did not include any representative of L. mediterranea which was described by Linaldeddu et al. [30] from several hosts, including V. vinifera in Italy. However, we have shown that L. vitis is phylogenetically indistinguishable from L. mediterranea. Their ITS sequences are 100% identical and the L. vitis tef1-α sequence deposited in GenBank differs from the tef1-α of L. mediterranea in 2 nt positions (1 missing G and a C instead of a T in the EF-986R primer binding region). We re-sequenced the tef1-α region of L. vitis CBS124060 (Table 2) using primers EF1-688F and EF1-1251R [35] which span a larger region than primers EF1-728F and EF-986R used by Yang et al. [40] and verified that these 2 nt are not real. There is no missing G in the L. vitis sequence and the C instead of a T is an artefact in L. mediterranea sequence introduced by the EF-986R primer sequence. Thus, the tef1-α region of L. vitis CBS124060 is 100% identical to the tef1-α sequence of L. mediterranea.

All the Botryosphaeriaceae isolates tested in the pathogenicity test were pathogenic to the citrus shoots. On the wood tissue under the bark, black to brown lesions developed, upward and downward from the inoculation point, within 30 days. The control plants did not develop any symptoms. Shapiro-Wilk test for normality revealed that the data differed significantly from a normal distribution (W = 0.83, p = 0.047). Lesion lengths varied between the species and among the isolates of each species tested, with a significant difference (F = 10.874; p < 0.001) (Table 3).

Table 3

Mean lesion lengths (cm) caused by Doth. viticola, D. mutila, D. seriata, L. mediterranea and L. mitidjana species implicated in citrus dieback in northern Algeria, 30 days after inoculation of detached green branches with mycelium-colonized agar plugs.

Species	Isolate code	Mean lesion length (cm) ± SD
D. seriata	ALG91	5.49±2.65 a
D. seriata	ALG98	2.35±0.55 cd
L. mediterranea	ALG36	4.39±1.31 ab
L. mediterranea	ALG40	3.83±0.97 abc
Doth. viticola	ALG86	2.82±0.86 bcd
Doth. viticola	ALG84	2.1±0.67 d
D. mutila	ALG102	2.04±0.54 d
D. mutila	ALG103	2.03±0.29 d
L. mitidjana	ALG39	3.88±1.24 abc
L. mitidjana	ALG34	2.2±0.67 cd

The same letter after numbers refers to the isolates that do not differ significantly according to Tukey’s HSD test at P ≤ 0.05.

The most aggressive isolates were ALG91 (D. seriata) and ALG36 (L. mediterranea), which produced the longest lesions (5.49±2.65 cm and 4.39±1.31 cm, respectively) with a statistically significant difference recorded between ALG91 and the rest of the species, except for L. mediterranea. No significant difference in lesions size was observed between the isolates ALG40 (L. mediterranea) and ALG39 (L. mitidjana), which presented intermediate lesion lengths (3.83±0.97 and 3.88±1.24 cm, respectively). However, the smallest lesion size was produced by Doth. viticola ALG84 with 2.1±0.67 cm and both D. mutila isolates ALG102 (2.04±0.54) and ALG103 (2.05±0.4 cm). D. seriata was the only species that showed significant difference in lesion length between its two isolates (Table 3).

Koch’s postulates were confirmed by a successful re-isolation of all tested fungal species from the necrotic tissues (Table 3).

Distribution of Botryosphaeriaceae species

Overall, the Botryosphaeriaceae species occurred in 42 of the 80 citrus trees showing canker and dieback symptoms (S2 Table). Five distinct Botryosphaeriaceae species were obtained in this study. Each species was found with its respective frequency, as follow: L. citricola (14.1%), L. mediterranea (13%) and D. seriata (10.9%), Doth. viticola (7.6%) and D. mutila (5.4%).

At least, two different species were found in each orchard. L. mediterranea and Doth. viticola were found in six of the ten surveyed orchards. They were followed by L. mitidjana, recorded from five orchards of two municipalities. D. seriata was found in four sampling sites; whereas, D. mutila was recovered from only two orchards of the same municipality.

Discussion

This study aimed to evaluate and characterize the diversity of the Botryosphaeriaceae species associated with dieback of Citrus sinensis. It represents the first survey and preliminary investigation of these species in the main citrus orchards in northern Algeria.

Citrus canker and dieback were detected in all regions surveyed. Several external symptoms, including partial or complete dieback of the tree, branch and shoot cankers were observed. Over time, the disease can increase and seriously affected trees can become barren and eventually, die. Similar situations have been described in several citrus orchards, worldwide [8, 17, 22, 23, 41]. According to previous studies, abiotic factors, including drought, severe sunburn or freezing predispose the trees to xylem dysfunction, leading to these diseases [3, 4, 41].

In this study, five species belonging to three different genera of the Botryosphaeriaceae were recovered from symptomatic citrus trees, namely: L. mediterranea, D. seriata, D. mutila, Doth. viticola and L. mitidjana. The latter is introduced here, as new species. To our knowledge, except for D. seriata and L. mediterranea, this is the first report of D. mutila, Doth. viticola and L. mitidjana, causing branch canker disease on citrus and any crop, in Algeria. The Botryosphaeriaceae species were recovered from more than half of the trees sampled and were found in all the prospected orchards.

Lasiodiplodia was the most commonly isolated genus that was found in six of the ten surveyed orchards. This fact is consistent with previous studies, which showed that Lasiodiplodia species have the ability to target a wide variety of plants, distributed worldwide [42-44]. In fact, Lasiodiplodia species do not only occur as latent endophytes in asymptomatic plants but are also associated with different symptoms occurring on a variety of hosts including stem-end rot, fruit rot, decline, canker and dieback [43, 45–47]. In this study, L. mediterranea and L. mitidjana sp. nov. were the most frequently encountered species. L. mediterranea has been reported as the causal agent of canker and dieback of grapevine, holm oak as well as citrus, indicating its capability to target different hosts [30]. The latter findings lead Andolfi et al. [48] to isolate and characterize the main secondary metabolites produced by L. mediterranea, as well as to evaluate its phytotoxic and antifungal activities. According to the former authors [30], L. mediterranea has been found only on V-shaped necrotic sectors of grapevine while, it has been isolated from all the lesion types of citrus trees in this study. L. mitidjana sp. nov. was found in five of the surveyed orchards. Isolates of this species were present predominantly in the wedge-shaped necrosis.

Doth. viticola was isolated at low frequency compared to Lasiodiplodia species found in this study. Interestingly, it was detected in six sampling sites. This species was first described as Spencermartinisia viticola by Phillips et al. [49]. It was obtained for the first time from Vitis vinifera in Spain. Recently, Yang et al. [40] regarded Spencermartinsia a synonym of Dothiorella and thus transferred the epithet viticola to Dothiorella as Doth. viticola. This taxonomic change was supported by a multi-gene phylogeny that included Spencermartinsia in Dothiorella genus [40, 50]. Doth. viticola has been reported from a wide range of woody hosts, including citrus trees [18, 23]. Recently, it has been also described as the causal agent of gummosis on citrus trees, in Tunisia [22]. According to Phillips et al. [20] and Dissanayake et al. [51], this species is known from China, Chile, USA, Spain, France, Australia, South Africa and Tunisia. Therefore, this study constitutes the first record of Doth. viticola in Algeria, which thus expands its known geographical range.

Two species of Diplodia genus, D. seriata and D. mutila were isolated from the surveyed orchards. Diplodia species are well known to cause damage on several economically important species and causing numerous disease symptoms including blight, dieback, rot diseases and canker [24, 30, 52–56]. In this study, D. seriata was frequently recovered from the sampling sites, which matches the findings of previous studies indicating the cosmopolitan nature of this species. This latter species is commonly reported as a pathogen on a large number of hosts and has been reported from hundreds of plant species [20, 51, 52]. D. mutila, the second Diplodia species isolated in this study, was less frequently found in the prospected orchards. Moreover, to our knowledge, this is the first report of this species in Algeria. In addition to Algeria, the USA is the only other country in which both D. seriata and D. mutila have been associated with citrus dieback [23]. These species have been found on apples in the USA [57, 58], Chile [59], France [60], Germany [61], Uruguay [62] and South Africa [63]; as well as in pear trees [62, 63], plum [64], peach and apricot [62, 65] and walnut [53].

All the Botryosphaeriaceae species of this study caused necrosis on the citrus shoots, with differences in the lengths of the lesions. These differences were observed between the species and also among isolates of the same species. According to the obtained results, the largest lesion was observed from one isolate of the D. seriata species (5.49 cm). Indeed, D. seriata was significantly different compared to the rest of the isolates, which is consistent with previous studies that showed significant impact of D. seriata on several hosts, across the globe [30, 66–68]. However, for this study, further tests need to be conducted in order to confirm our findings. According to the data from Table 3, considering the species aggressiveness, we could say that L. mediterranea is the most aggressive due to the size of both isolates tested in this study, with lesion lengths of 4.39 cm and 3.83 cm respectively.

For L. mediterranea, our results are in accordance with a previous study, which highlighted its aggressiveness in artificial inoculation experiments [30]. The smallest lesions were obtained from the isolates of D. mutila and Doth. viticola. Nevertheless, this was not the case in another study comparing D. mutila to L. theobromae and D. seriata, in which D mutila was found to have the largest lesions length [62]. According to Linaldeddu et al. [30] and Chakusary et al. [52], these differences in aggressiveness maybe due to several factors including genetic variability of isolates, age, type of host tissue, differences in susceptibility as well as inoculation methods and experimental conditions. In this case, extensive sampling from citrus as well as other hosts are required to further emphasise the findings and draw a final solid conclusion.

Overall, almost all the Botryosphaeriaceae species we identified have previously been detected on citrus trees with the exception of L. mitidjana, which was described for the first time associated with citrus dieback. Given the major impact of the Botryosphaeriaceae species isolated on declining trees, worldwide, it is important to emphasize the urgent need to implement prevention techniques and management strategies in order to minimize the incidence of these pathogens and to prevent their spread to new orchards. For a better understanding of citrus dieback, it is necessary to set up larger surveys that include all citrus production areas. These surveys would assess, more accurately, the impact of the trunk diseases pathogens and eventually identify the factors that influence the dieback. This will be set in order to identify a number of practices to prevent their development.

Maximum Parsimony phylogenetic tree resulting from the analysis of the combined ITS and tef1-α sequence data from Lasiodiplodia species.

The tree was rooted to Diplodia mutila and Diplodia seriata.

(TIF)

Click here for additional data file.

Details of strains included in the phylogenetic and/or morphological analyses.

(DOCX)

Click here for additional data file.

Distribution of the Botryosphaeriaceae species among the surveyed orchards.

(DOCX)

Click here for additional data file.

4 Dec 2019

PONE-D-19-29810

Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria

PLOS ONE

Dear Dr. BERRAF-TEBBAL,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We have now obtained two thoughtful and very complete reports from external reviewers on study PONE-D-19-29810 “Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria”. Both reviewers and myself agree that this work addresses interesting questions as still very little is known about Botryosphaeriaceae in Algeria.  The study is well presented and the reviewers are generally positive about the results, however, they have also identified some major concerns. Based on the reviews and my own assessment, I am recommending that the authors should resubmit a revised version of the paper that takes note of all the reviewers' comments. They should also include a detailed letter describing how the concerns have been taken into account. In their revision, the authors should not overstate what can be concluded from the results and, therefore, add caveats where necessary.

Please also verify that all the data generated for this study has been made available. Reviewer 1 mentioned that the GenBank accession for the Tef1-α sequences was not available online.

Some additional minor remarks:

L.273 ..we have shown…

L. 279 “…these 2 nt are not real.” Are not real is confusing, maybe instead use “ were correctly determined”?

L. 335 “The remaining species…” better mention the species’ name here.

L. 351 A similar situation has been…

L. 363 worLd

L. 373 compared instead of comparing

L. 389 “…known for targeting economically important plants…” targeting sounds as if they are selecting the hosts based on the economical value. Maybe better say: …known to cause damage on several economically important species...”

l. 393 “The later…”, better say “This latter species..” or just “It...”

L. 408 …with a previous study…

I would suggest to add some research perspectives at the end of the discussion. What kind of studies could help to better understand and develop management recommendations against citrus dieback?

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Reviewer #1: General comments:

Akila Berraf-Tebbal and collaborators present in this manuscript the results of a survey on Botryosphaeriaceae diversity and pathogenicity affecting symptomatic sweet orange (Citrus sinensis) in Algeria. Botryosphaeriacea species were identified in every orchard tested (n=10) and a total of five different species have been isolated from symptomatic samples, with frequencies ranging from 5.4% to 14.1% of the samples. One species appears to form a distinct monophyletic group - not described before - and is claimed by the authors to be a new species. The pathogenicity of two representative isolates for every species isolated (n=5) was tested experimentally on Citrus shoot and Koch’s postulate was verified for all of them. Differences in pathogenicity was observed between isolates.

Overall, the manuscript is well written and there is no doubt about the efforts made by the authors to produce this work. The results presented are interesting and alert about the potential spread and threats brought by these pathogens, as mentioned by the authors.

My main comments to improve this manuscript is that authors often goes on conclusions that are not completely supported by the results. I suggest therefore to reconsider these conclusions or change the way they are presented to stick more on the facts. Furthermore, the GenBank accession for the Tef1-α sequences are not available which hamper my reviewing conclusions. This is even more problematic because the genetic difference observed to discriminate the new species are brought by this marker.

In this sense, a new species (Lasiodiplodia mitidja) is introduced in this study. This introduction is based on a two loci phylogeny, as well as morphological observations. I’m not a taxonomist myself, but are two SNPs (which I could not verified, and that is not illustrated by an alignment neither in the manuscript) and a bootstrap of 80 enough to consider the organism a new species? Concerning the morphological differences, as the authors mentioned, conidia “tend” to be larger and L/W ratio is different but for both measurements, no statistical significance is brought to the observations to confirm the difference. Can it represent a subpopulation of L. citricola? I presume it is not possible to test if those “species” outcross but if we have to be more rigorous, I would recommend to stay more prudent about the “new species” terminology and presented it more as a suggestion, or inform the readers that all the criteria to say it’s a new species are not completely fulfil.

My second concern is the ambiguity made between the types of wood alteration/symptoms observed in Citrus trees and the presence and implication of Botryosphaeriaceae. Botryosphaeriaceae species can be isolated from certain types of alteration and yet not being responsible of these alterations. Knowing the “opportunist” behaviour of these pathogens, I would not be surprised if they take over the habitat after a disequilibrium was induced into the microbiome of trees following another pathogen attack. The fact that the isolates were able to provoke symptoms experimentally does not necessarily mean they are responsible of the ones observed on the diseased trees, especially since the symptoms observed after artificial inoculation are not correlated to the ones observed on fields. Similarly, the presence of basidiocarps on heavily symptomatic Citrus is confusing for me, at least the way it is presented. What is the link between the Botryosphaeriaceae and the basidiocarps emergence, which species correspond to this basidiocarp? In a similar fashion, Figure 4 is confusing as my conclusion on this figure is that Botryosphaeriaceae can be isolated from different types of symptoms and not that one species is more isolated from one type of symptoms than another as the authors tend to say. There is no statistic proving so, and a quick interpretation (but false) from a hurried reader would be that such species is responsible of such symptoms. At this point, those results are more detrimental that beneficial to the study. I either recommend to delete this part or erase those ambiguities by a deeper discussion and a clearer result presentation. What do we know about the multifactorial aspects of dieback diseases? Is there only one pathogen involved? I think study conducted on Botryosphaeriaceae and grapevine trunk disease can be related to this case. Furthermore, if this part is conserved, more insights on what is known about the different symptoms that can cause Botryosphaeriaceae could/should be presented in the introduction.

Finally, the statistical methods used to test pathogenicity differences is either not well presented or the conclusions are not correct. This part needs to be improve. Have you tested species effect? Isolate effect? What are the p-value attributed to each ANOVA test, which factor has been tested by the ANOVA? Is LSD method (which is not described by the way) the more appropriate in your case?

Minor Comments:

L30: 14.1% percent of the samples and 13% of the samples

L31: what is the difference between widespread and abundant?

L42: I would erase (pomelos)

L43: Despite the high adaptation capacity of citrus trees to different climates (reference is missing)

L47: Citrus diseases are numerous and diverse, and are caused by phytopathogenic agents belonging to viruses, viroids, phytoplasmas, bacteria, and fungi (reference is missing).

L57: reference is missing

L63: colonize or affect?

This part on Botryosphaeriaceae should be more documented: classification of Botryosphaeriaceae, how many genera, endophytes with symptomless period, etc…

L82: Surveys were conducted in ten commercial orchards in the northern region of Algeria, specifically, in the Mitidja plain at the base of the Tell Atlas Mountains (Table 1).

Table 1: I would add coordinates of the orchards and years of sampling.

L94: was the scalpel sterilized?

L112: In this paragraph, can you add more infos about the PCR conditions?

L120-121: pyrosequencing?

L123: Newly generated sequences were deposited in GenBank (Table 2): the Tef1-α isn’t accessible.

L124: Sequences for both DNA regions were retrieved in BLAST searches from GenBank [34].

Check the meaning of this sentence. For example: “Homological sequences of the newly sequenced ones were retrieved from the GenBank by Blast.”

L125: Table 2: Can you add more info on this table, like type of tissue (trunk/branches), type of symptoms (under your classification), Orchard, etc….

L132: Please specify the request you made, or put the sequence in the supplemental files.

L136: what kind of adjustments?

L153: diameter

L164: this part need to be more precise/improve. Which threshold to accept the significance of the ANOVA, which factor tested, what LSD means for, which soft did you use?

L171: nothing is said about the distribution

L172-173 and L174-175 could be fused for clarity purposes

L175: the total number of samples… Samples = branches?

L176 – 177: this measurement is completely arbitrary and according to me abusive. If one pathogen would have occurred at 80% frequency, the difference between 11%(very frequent) and 4%(infrequent) would be meaningless. My advice, stick to the numbers and do not try to interpreted it in a frequent or infrequent way, that’s too subjective.

L186: On heavily infected trees, basidiocarps emerged: that’s ambiguous, as said before.

L189: why wedge-shaped necrosis is not name WSN?

L191: similar BCN instead of NBC and YSW instead of NCC.

L196: the “e.” is missing on the picture, but maybe see in it as a sign for not putting this picture…

L214: Why MP tree is not shown? At least in supplemental file?

L218: why 23 isolates and not 24 (10+14)

L228: The phylogenetic tree of only one method is presented, although the bootstrap values of the two methods are shown. Can we see the tree constructed with the second method in the supplemental files?

L279: can you have the sequence accessible please?

L311: from what I’ve read, LSD test is not recommended anymore as sensitive to multiple comparison. Furthermore, as mentioned above, this test is not well conducted and presented. A histogram would be better, with a sign for significant difference, either at the isolate level and species level, with threshold use for significance. The 100% of re-isolation frequency for every isolates are not necessary in the table according to me, if you say it in the text.

L317: Ambiguous: are you speaking of distribution in the wood? If yes, cut this paragraph in two: Frequency of occurrence / Distribution of Botryosphaeriaceae in the wood.

L320: I comment already the frequent and very frequent ranking, that’s abusive according to me.

L323: For each orchard, at least two different species were isolated, average per orchard?

L328: new paragraph or no paragraph at all as mentioned above, I found this part confusing.

L370-371: According to these authors, L. mediterranea has been found only on V-shaped necrotic sectors of grapevine while it has been isolated from all the lesion types of citrus trees in this study.

L373: what the results of Andolfi et al. bring to your results?

L374-375: maybe if you had isolated only the 4 or 5 isolates that were from brown central necrosis, would you have said that the species was exclusively found in brown central necrosis? This part of the discussion is not really constructive.

L387: More interesting that this, it confirms its wide geographical range.

L402: Wedge shaped lesion?

L403-405: this part of the discussion goes beyond what your data show, either improve your statistical analysis or moderate the message.

L408-415: similar, hard to have this kind of discussion with two isolates per strains, with one phenotyping trial.

L446: References have to be reformatted: species not in italic, capital letter on every first word letter for some references, etc.; some other problem like L508 Phililips is written bizarrely.

Reviewer #2: This is a nice study of Botryosphaeriaceae which are important plant pathogens, including fruit trees. Algeria is unexplored both from mycological and pathological perspective and it is nice to see collaborations like this resulting in a good piece of work. I agree with authors regarding Lasiodiplodia mediterranea and L.vitis situation, especially because pcr atifacts introduced by primer sequences are unfortunately a common thing these days (my personal experience). I have small suggestions that would improve the paper.

Abstract, line 25-of Botryosphaeriaeceae

Abstract, line 29- Delete which, add Lasiodiplodia mithidjana is described in this paper as a new species

Abstract, line 62- delete effect

Materials-line 98-dried on sterilized paper (towels, filter paper?)

Materials, line 100-The mycelium emerging from wood pieces was transferred…

Materials, line 105-Isolates that lacked pycnidia production…

Materials, line 150-How did you select representative isolates?

Materials, line 151-Shoots? But above you mentioned branches (line 148)

Materials, line 157-“… well watered and maintained under favorable conditions” What do you mean by this? Were the cuttings in soil or in water? How many times per week did you change the water? Ambient temperature? Light?

Results, line 182-Degrees of intensity? Where are they?

Results, line 186-Basidiocarps of which species or genera?

Results, line 309-What about control plants?

Results, line 313-Now you mention branches again

Discussion, line 360-and seriously affected trees can become

Discussion, lines 403-404- “However, D. seriata was significantly different compared to the rest of the isolates” But previously you said that both D. seriata and L. mediterranea were most aggressive species (based on lesion lengths). So which species was in fact most aggressive? Also, what about differences in aggressiveness between different isolates of the same species?

Discussion, lines 408-412-Was this previous study also about Bot on citrus trees?

Discussion, lines 409 and 411-In line 404 you are talking about aggressiveness. Now about virulence. In line 413 you talk again about aggressiveness. Virulence and aggressiveness don’t mean the same thing. Replace the term virulence with aggressiveness in lines 409 and 411.

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Reviewer #1: Yes: Benoit LAURENT

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19 Jan 2020

Answers to the reviewer 1

General comments:

Akila Berraf-Tebbal and collaborators present in this manuscript the results of a survey on Botryosphaeriaceae diversity and pathogenicity affecting symptomatic sweet orange (Citrus sinensis) in Algeria. Botryosphaeriacea species were identified in every orchard tested (n=10) and a total of five different species have been isolated from symptomatic samples, with frequencies ranging from 5.4% to 14.1% of the samples. One species appears to form a distinct monophyletic group - not described before - and is claimed by the authors to be a new species. The pathogenicity of two representative isolates for every species isolated (n=5) was tested experimentally on Citrus shoot and Koch’s postulate was verified for all of them. Differences in pathogenicity was observed between isolates.

Overall, the manuscript is well written and there is no doubt about the efforts made by the authors to produce this work. The results presented are interesting and alert about the potential spread and threats brought by these pathogens, as mentioned by the authors.

My main comments to improve this manuscript is that authors often goes on conclusions that are not completely supported by the results. I suggest therefore to reconsider these conclusions or change the way they are presented to stick more on the facts.

We took into consideration all the comments regarding the conclusions and we made sure to do all the required modifications.

Furthermore, the GenBank accession for the Tef1-α sequences are not available which hamper my reviewing conclusions. This is even more problematic because the genetic difference observed to discriminate the new species are brought by this marker.

The ITS and the tef1-α sequences have been deposited into GenBank; However, the tef1-α sequences are not automatically deposited into GenBank after being accessioned. Each sequence record is individually examined and processed by the GenBank annotation staff to ensure that it is free of errors or problems.

In this sense, a new species (Lasiodiplodia mitidja) is introduced in this study. This introduction is based on a two loci phylogeny, as well as morphological observations. I’m not a taxonomist myself, but are two SNPs (which I could not verified, and that is not illustrated by an alignment neither in the manuscript) and a bootstrap of 80 enough to consider the organism a new species? Concerning the morphological differences, as the authors mentioned, conidia “tend” to be larger and L/W ratio is different but for both measurements, no statistical significance is brought to the observations to confirm the difference. Can it represent a subpopulation of L. citricola? I presume it is not possible to test if those “species” outcross but if we have to be more rigorous, I would recommend to stay more prudent about the “new species” terminology and presented it more as a suggestion, or inform the readers that all the criteria to say it’s a new species are not completely fulfil.

We agree that this may be debatable. In fact, we have discussed this previously within the team. However, it is clearly aligned with the current trend for introduction of novel Lasiodiplodia species.

In the future it may well be proven that in fact it is not a new species different from L. citricola. But taxonomy is dynamic and hence frequently changing. For the moment we would like to introduce the new species. The fact is that eventually it will be described as a new species, if not by our group, then by someone else.

As an example of a case which is similar to ours, here are the nucleotide differences for the following mentioned species: L. chinensis vs L. lignicola vs L. pseudotheobromae. For all 3 species ITS is 100 % identical

As for Tef1:

L. chinensis vs L. lignicola: 1 nucleotide difference

L. chinensis vs L. pseudotheobromae: 3 nucleotides differences

My second concern is the ambiguity made between the types of wood alteration/symptoms observed in Citrus trees and the presence and implication of Botryosphaeriaceae. Botryosphaeriaceae species can be isolated from certain types of alteration and yet not being responsible of these alterations. Knowing the “opportunist” behaviour of these pathogens, I would not be surprised if they take over the habitat after a disequilibrium was induced into the microbiome of trees following another pathogen attack.

We agree with the reviewer. After considering your other comments on the same part and your suggestion of removing it and given that it is not relevant for the paper we decided to delete it.

The fact that the isolates were able to provoke symptoms experimentally does not necessarily mean they are responsible of the ones observed on the diseased trees, especially since the symptoms observed after artificial inoculation are not correlated to the ones observed on fields.

We agree with the reviewer, however the goal was to test pathogenicity of the isolates and this is the way to do it. Of course we cannot be sure that they will behave the same way in the field but they have the potential to do so.

Similarly, the presence of basidiocarps on heavily symptomatic Citrus is confusing for me, at least the way it is presented. What is the link between the Botryosphaeriaceae and the basidiocarps emergence, which species correspond to this basidiocarp?

We described the health status of the orchards where the sampling has been carried out (branch and shoot cankers, abnormal growth of epicormic shoots; defoliation and leaf chlorosis). Basidiocarps are the fruiting bodies of the decay-causing fungi. Their presence on the trunk means that it is an already rotting trunk and that some ascomycetes (Botryosphaeriaceae, Diatrypaceae…) and Basidiomycetes (Fomitiporia, Phellinus….) have already colonized the trunk.

In a similar fashion, Figure 4 is confusing as my conclusion on this figure is that Botryosphaeriaceae can be isolated from different types of symptoms and not that one species is more isolated from one type of symptoms than another as the authors tend to say. There is no statistic proving so, and a quick interpretation (but false) from a hurried reader would be that such species is responsible of such symptoms. At this point, those results are more detrimental that beneficial to the study. I either recommend to delete this part or erase those ambiguities by a deeper discussion and a clearer result presentation. What do we know about the multifactorial aspects of dieback diseases? Is there only one pathogen involved? I think study conducted on Botryosphaeriaceae and grapevine trunk disease can be related to this case. Furthermore, if this part is conserved, more insights on what is known about the different symptoms that can cause Botryosphaeriaceae could/should be presented in the introduction.

We agree with the reviewer and his comments. After pointing out these remarks we thoughtfully considered them and decided to delete the figure 4 as well as the paragraphs related to it.

Finally, the statistical methods used to test pathogenicity differences is either not well presented or the conclusions are not correct. This part needs to be improve. Have you tested species effect? Isolate effect? What are the p-value attributed to each ANOVA test, which factor has been tested by the ANOVA? Is LSD method (which is not described by the way) the more appropriate in your case?

We took into consideration your valuable comments and we made sure to change this part and we removed all the ambiguities.

Minor Comments:

L30: 14.1% percent of the samples and 13% of the samples

R: Revised as recommended

L31: what is the difference between widespread and abundant?

R: Widespread means that it is found or distributed over a large area. However, abundant means that it is existing or available in large quantities (it could have the same meaning as plentiful)

L42: I would erase (pomelos)

R: Revised as recommended

L43: Despite the high adaptation capacity of citrus trees to different climates (reference is missing)

R: Revised as recommended

L47: Citrus diseases are numerous and diverse, and are caused by phytopathogenic agents belonging to viruses, viroids, phytoplasmas, bacteria, and fungi (reference is missing).

R: Revised as recommended

L57: reference is missing

R: Revised as recommended

L63: colonize or affect?

R: We deleted ‘affect’

This part on Botryosphaeriaceae should be more documented: classification of Botryosphaeriaceae, how many genera, endophytes with symptomless period, etc…

R: Revised as recommended

L82: Surveys were conducted in ten commercial orchards in the northern region of Algeria, specifically, in the Mitidja plain at the base of the Tell Atlas Mountains (Table 1).

Table 1: I would add coordinates of the orchards and years of sampling.

R: Revised as recommended

L94: was the scalpel sterilized?

R: Yes, the scalpel was sterilized. This detail has been added to the manuscript.

L112: In this paragraph, can you add more infos about the PCR conditions?

R: Revised as recommended

L120-121: pyrosequencing?

R: the company used Sanger sequencing method.

L123: Newly generated sequences were deposited in GenBank (Table 2): the Tef1-α isn’t accessible.

R: The sequences are available in GenBank.

L124: Sequences for both DNA regions were retrieved in BLAST searches from GenBank [34].

Check the meaning of this sentence. For example: “Homological sequences of the newly sequenced ones were retrieved from the GenBank by Blast.”

R: Revised as recommended

L125: Table 2: Can you add more info on this table, like type of tissue (trunk/branches), type of symptoms (under your classification), Orchard, etc….

R: Revised as recommended

L132: Please specify the request you made, or put the sequence in the supplemental files.

R: Revised as recommended (the sequences are in the supplementary files)

L136: what kind of adjustments?

R: The ITS and tef1-α sequences were initially aligned separately using ClustalX v. 1.83. The alignments were manually optimized by coding the missing sequences as “?”. Ambiguous sequences at the start and the end were deleted and gaps were adjusted in BioEdit.

L153: diameter

R: Revised as recommended

L164: this part need to be more precise/improve. Which threshold to accept the significance of the ANOVA, which factor tested, what LSD means for, which soft did you use?

R: Revised as recommended

Detailed responses:

Which threshold to accept the significance of the ANOVA

R: when the P value is below the threshold (0.05), the difference between the means is considered as significant.

Which factor tested?

R: We tested the lesions produced by each fungal isolate of the different species.

What LSD means for?

R: We changed the statistical test by using Tukey's honestly significant difference (HSD) test.

Which soft did you use?

R: The R v. 3.5.1 statistical software was used to perform the statistical analysis.

L171: nothing is said about the distribution

R: We removed the word ‘distribution’ from the title

L172-173 and L174-175 could be fused for clarity purposes

R: We removed this paragraph as recommended.

L175: the total number of samples… Samples = branches?

R: The samples mean the different necrotic lesions found in the branches and the trunks of the 80 trees.

L176 – 177: this measurement is completely arbitrary and according to me abusive. If one pathogen would have occurred at 80% frequency, the difference between 11%(very frequent) and 4%(infrequent) would be meaningless. My advice, stick to the numbers and do not try to interpreted it in a frequent or infrequent way, that’s too subjective.

R: We removed the paragraph related to the frequency of occurrence, as recommended.

L186: On heavily infected trees, basidiocarps emerged: that’s ambiguous, as said before.

R: We removed the description of the basidiocarps from the photoplate as well as from the text, as recommended.

L189: why wedge-shaped necrosis is not name WSN?

R: Revised as recommended

L191: similar BCN instead of BCN and YSW instead of NCC.

R: Revised as recommended

L196: the “e.” is missing on the picture, but maybe see in it as a sign for not putting this picture…

R: Revised as recommended

L214: Why MP tree is not shown? At least in supplemental file?

R: Revised as recommended (the tree is in supplementary file)

L218: why 23 isolates and not 24 (10+14)

R: The sequence of one isolate was not good enough to use it for the phylogenetic analysis.

L228: The phylogenetic tree of only one method is presented, although the bootstrap values of the two methods are shown. Can we see the tree constructed with the second method in the supplemental files?

R: Revised as recommended

L279: can you have the sequence accessible please?

R: The sequence has been submitted to GenBank. It will be available online after verification of the annotation. We have included the accession number into the table 2.

L311: from what I’ve read, LSD test is not recommended anymore as sensitive to multiple comparison. Furthermore, as mentioned above, this test is not well conducted and presented. A histogram would be better, with a sign for significant difference, either at the isolate level and species level, with threshold use for significance. The 100% of re-isolation frequency for every isolates are not necessary in the table according to me, if you say it in the text.

R: Revised as recommended.

L317: Ambiguous: are you speaking of distribution in the wood? If yes, cut this paragraph in two: Frequency of occurrence / Distribution of Botryosphaeriaceae in the wood.

R: Revised as recommended

L320: I comment already the frequent and very frequent ranking, that’s abusive according to me.

R: We deleted this paragraph.

L323: For each orchard, at least two different species were isolated, average per orchard?

R: In the paragraph L323, we described the widespread of the species in the orchards. It was only to mention their presence on each orchard.

For more details, here is a table containing all the information about species distribution among the surveyed orchards.

Region

Oued El Alleug Chiffa Boufarik Staoueli

Species/ Orchards 1 2 3 4 5 6 7 8 9 10 Total

D. seriata - - - - 2 1 - - 3 4 10

D. mutila - - - - 3 2 - - - - 5

L. mediterr. 2 2 3 1 - - 2 2 - - 12

L. mitidjana 0 3 3 3 - - 2 2 - - 13

Doth. vitic. 1 - 1 - 1 2 - - 1 1 7

Total 3 5 7 4 6 5 4 4 4 5 47

L328: new paragraph or no paragraph at all as mentioned above, I found this part confusing.

R: Revised as recommended (We removed the paragraph).

L370-371: According to these authors, L. mediterranea has been found only on V-shaped necrotic sectors of grapevine while it has been isolated from all the lesion types of citrus trees in this study.

R: Revised as recommended

L373: what the results of Andolfi et al. bring to your results?

R: Andolfi et al. (2016) isolated and characterized the main secondary metabolites produced by L. mediterranea. They also, evaluated its phytotoxic and antifungal activities. These findings support our results, which show the ability of this fungal species to colonize and cause damages in the wood.

L374-375: maybe if you had isolated only the 4 or 5 isolates that were from brown central necrosis, would you have said that the species was exclusively found in brown central necrosis? This part of the discussion is not really constructive.

R: we removed the paragraph, as well as the figure 4.

L387: More interesting that this, it confirms its wide geographical range.

R: revised as recommended

L402: Wedge shaped lesion?

R: The paragraph is about the pathogenicity trial and the lesions produced by each Botryosphaeriaceae species. We did not consider the shape of the lesions, for the pathogenicity test.

L403-405: this part of the discussion goes beyond what your data show, either improve your statistical analysis or moderate the message.

R: Revised as recommended

L408-415: similar, hard to have this kind of discussion with two isolates per strains, with one phenotyping trial.

R: Revised as recommended

L446: References have to be reformatted: species not in italic, capital letter on every first word letter for some references, etc.; some other problem like L508 Phililips is written bizarrely.

R: Revised as recommended

Answers to the reviewer 2

Reviewer #2: This is a nice study of Botryosphaeriaceae which are important plant pathogens, including fruit trees. Algeria is unexplored both from mycological and pathological perspective and it is nice to see collaborations like this resulting in a good piece of work. I agree with authors regarding Lasiodiplodia mediterranea and L.vitis situation, especially because pcr atifacts introduced by primer sequences are unfortunately a common thing these days (my personal experience). I have small suggestions that would improve the paper.

Abstract, line 25-of Botryosphaeriaeceae

R: Revised as recommended

Abstract, line 29- Delete which, add Lasiodiplodia mitidjana is described in this paper as a new species

R: Revised as recommended

Abstract, line 62- delete effect

R: Revised as recommended

Materials-line 98-dried on sterilized paper (towels, filter paper?)

R: Revised as recommended

Materials, line 100-The mycelium emerging from wood pieces was transferred…

R: Revised as recommended

Materials, line 105-Isolates that lacked pycnidia production…

R: Revised as recommended

Materials, line 150-How did you select representative isolates?

R: We selected two isolates, from each phylogenetically resolved species.

Materials, line 151-Shoots? But above you mentioned branches (line 148)

R: Revised as recommended (We have standardized using shoot instead of branch).

Materials, line 157-“… well watered and maintained under favorable conditions” What do you mean by this? Were the cuttings in soil or in water? How many times per week did you change the water? Ambient temperature? Light?

R: The inoculated cuttings were wrapped with wet sterile cotton to avoid the desiccation of the agar plug. The shoots were immediately transplanted into pots containing sterilized water as a growth substrate (10 shoots per pot), which were incubated at the ambient room temperature, under daily photoperiod. The water of the container was changed twice a week.

Results, line 182-Degrees of intensity? Where are they?

R: The degrees of intensity refer to the different levels of the dieback symptoms observed in the orchards.

Results, line 186-Basidiocarps of which species or genera?

R: We did not identify the basidiocarps. We described all the symptoms related to the citrus trees dieback, including the fruiting bodies emerged from the trunks.

Results, line 309-What about control plants?

R: We did not isolate any of the tested species from the negative control.

Results, line 313-Now you mention branches again

R: revised as recommended

Discussion, line 360-and seriously affected trees can become

R: revised as recommended

Discussion, lines 403-404- “However, D. seriata was significantly different compared to the rest of the isolates” But previously you said that both D. seriata and L. mediterranea were most aggressive species (based on lesion lengths). So which species was in fact most aggressive? Also, what about differences in aggressiveness between different isolates of the same species?

So which species was in fact most aggressive?

R: The significant difference was made based on a comparison between all the tested isolates. It was not about the pairwise comparisons that take one isolate and compare it with each of the rest of isolates. D. seriata and L. mediterranea were the most aggressive species when compared to the rest of the species. However, D. seriata was the most aggressive species, considering the length of the lesion for each isolate, separately.

Also, what about differences in aggressiveness between different isolates of the same species?

R: Significant variation in aggressiveness can occur within and among isolates from the same species. This aggressiveness refers to the quantitative variation of pathogenicity on the susceptible host infection efficiency, the latent period, the spore production rate and the infectious period of each strain. These components are closely related to the genetic variability within the strains of the same species.

Discussion, lines 408-412-Was this previous study also about Bot on citrus trees?

R: the study was about Lasiodiplodia species (Botryosphaeriaceae) on grapevine.

Discussion, lines 409 and 411-In line 404 you are talking about aggressiveness. Now about virulence. In line 413 you talk again about aggressiveness. Virulence and aggressiveness don’t mean the same thing. Replace the term virulence with aggressiveness in lines 409 and 411.

R: Revised as recommended

Answers to the academic editor

L.273 ..we have shown…

R: Revised as recommended

L. 279 “…these 2 nt are not real.” Are not real is confusing, maybe instead use “ were correctly determined”?

R: Revised as recommended

L. 335 “The remaining species…” better mention the species’ name here.

R: Revised as recommended

L. 351 A similar situation has been…

R: Revised as recommended

L. 363 worLd

R: Revised as recommended

L. 373 compared instead of comparing

R: Revised as recommended

L. 389 “…known for targeting economically important plants…” targeting sounds as if they are selecting the hosts based on the economical value. Maybe better say: …known to cause damage on several economically important species...”

R: Revised as recommended

l. 393 “The later…”, better say “This latter species..” or just “It...”

R: Revised as recommended

L. 408 …with a previous study…

R: Revised as recommended

I would suggest to add some research perspectives at the end of the discussion. What kind of studies could help to better understand and develop management recommendations against citrus dieback?

R: Revised as recommended

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

17 Mar 2020

PONE-D-19-29810R1

Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria

PLOS ONE

Dear Dr. BERRAF-TEBBAL,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We have now received positive evaluations of manuscript PONE-D-19-29810 by two thoughtful reviewers who still ask for some mostly minor changes. I have read and evaluated the manuscript myself and agree with the reviewers. In the following, I will also provide some comments that might help to further improve the manuscript and make it more attractive to a broader readership.

I recommend starting out the introduction with a wider scope. Citrus cultivation is not only important in Algeria but in many countries and the fruits are being exported and eaten in even more countries, therefore I would start out with some sentences about the importance of Citrus in general. This could make the publication more attractive to a wider readership.

The authors mention at the end of the introduction that Botryosphaeriaceae have not been studied in detail in Algeria. Have other pathogen families or groups on Citrus already been studied in more detail? I think this should be introduced here in detail! This could also explain why the authors only focus on Botryosphaeriaceae and not check which other pathogens are causing the symptoms in their samples. Please explain that in more detail!

The objectives at the end of the introduction should be described in more detail! The authors do not even mention that they also aimed at testing the aggressiveness of the the isolates!

Table 1, I agree with Benoit Laurent that it would be relevant (if possible) to add the GPS coordinates of each orchard in each region as it would give an idea of how far apart the orchards are. If this information cannot be given, I would rather remove the GPS coordinates of the cities.

l. 145 Mention which was the best fitting DNA evolution model.

Please improve the description of the statistical analyses and test specifically for differences in aggressiveness between species as suggested by Benoit Laurent.

I am a little surprised about the paragraph called “Nomenclature”. I am not familiar with the description of new fungal species but I have never seen such a justification. Is this typical? Otherwise, it should be removed. I suggest to name this paragraph “Taxonomy” which should be mentioned that it has been described in detail and that the new names have been submitted to MycoBank.

In the results the authors mention that they obtained 47 fungal colonies belonging to Botryosphaeriaceae. Did they also observe other fungi in their isolates? What happened to the other samples?

l. 41 clementines

l. 62-64 This sentence is unclear. Do the authors refer to a single species of the Botryosphaeriaceae family or several that are all important pathogens? Please clarify!

l. 77 …citrus trees in Algeria…

l. 350 …similar situations have been described…

l. 351 …according to some authors… is not a good formulation!

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

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Reviewer #1: Partly

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I would like to thank Akila Berraf-Tebbal and his collaborators to the answers they brought to my questions and comments. The data on the tef1 alpha are now available, which was one major concern of my last review, and allow me to verify the monophyletic group corresponding to the introduction of a new species. The debate on this introduction is still questionable but does not question the quality and the presentation of the work. I don’t think that following a trend is really constructive and yet scientific, but I endorse their point of view about the dynamicity of the taxonomy. Besides, I’m rather surprised that, in response to my comment about the lack of general information on Botryosphaeriaceae, a sentence is brought from a reference and citing “It comprises 24 genera encompassing 222 species”, which is a rather static way of introducing the phylogeny of Botryosphaeriaceae. I would rather say “It comprises at least 24 genera encompassing 222 species”, or “It comprises at least 24 known genera encompassing 222 known species”.

Overall, I find the manuscript clearer as it is and have only one major suggestion to make this manuscript in the standard for publication. It concerns the pathogenicity test, the statistical analysis conducted on it and its interpretation. First of all, I suggest to add in the material and method (Pathogenicity test section) that lesion size was used as a proxy to measure the aggressiveness of the isolate. As you mentioned to one of the other reviewer comment, aggressiveness is a complex trait and proxies are often used, and are always good to define in the text. Then, the "Statistical analyzes" section from the Materials and Methods has still a margin of improvement. In your case, the ANOVA was used to test the genotype effect on the lesion size variation, with significance accepted for P<0.05. The test used to test the normality of the data could be also presented. Finally, what you can say so far is that some isolates belonging to some species was more aggressive than other isolates, belonging maybe to other species. But, you didn’t test the species effect, which would appreciate the variation of the means per species and test if the means are different. Hence you cannot say that one species is more aggressive than another one (L403 in the discussion, and the whole paragraph suggest so as it is presented). If the standard deviation is too important (D. seriata for example), therefore you’ll need more genotype per species to reach statistical significance and confirm those species effects, and then used HSD to identify which species is statistically different from the others. Therefore, I suggest to adapt the discussion in consequence, or add more genotypes in the pathogenicity test if possible.

Finally, I hope this section will be more accurately presented and this manuscript will be published as the results from this work will be beneficial for the community.

Minor comments:

I’m surprised by the answer of one of my comment about the basidiocarp observed in some citrus orchard:

“Basidiocarps are the fruiting bodies of the decay-causing fungi. Their presence on the trunk means

that it is an already rotting trunk and that some ascomycetes (Botryosphaeriaceae, Diatrypaceae…)

and Basidiomycetes (Fomitiporia, Phellinus….) have already colonized the trunk.”

Is that always true? For me, some decay-causing fungi are able to provoke the rotting of the trunk by

themselves (e.g. Heterobasidion on pine trees).

L95: There may have a misunderstanding in my comment. I was more interested in the GPS location of the orchards than the GPS location of the city. If the authors don’t have access to this information, this is not a big deal. The aim of my comment was to give additional information that could feed the discussion about the distribution pattern of the Bot species. Was L. mitidjana sp. nov. isolated both year? In similar orchard (tree ages?)

L374: “L. mitidjana sp. nov. was found in the five surveyed orchards. “ suggests that L. mitidjana sp. nov. was isolated from all the surveyed orchards. I suggest “L. mitidjana sp. nov. was found on five orchards out of ten”, or “L. mitidjana sp. nov. was found in five of the surveyed orchards”. Furthermore, do you have some elements to discuss the distribution of the species? Is there any particularity on these locations? The table containing all the information about species distribution and presented in response to my comment is particularly interesting. I’m surprised this table is not presented, at least in supplemental file.

L397: “our country” is maybe to personal

Reviewer #3: Dear Authors

I found this piece of work really interesting, well done, with a very good language, robust methodology and, much important, the article has been strongly improved after the first revisions.

A very few small notes are included in the attached pdf.

Furthermore, my only big concern, as mentioned by another reviewer, is about the description of the new Lasiodiplodia species. But I have read what you authors already have replied to the first reviewer and I can understand your point of view.

So, I accept your reasons and your determination to consider this as a novel species.

Probably, just to be clearer in the manuscript, it could be useful to sequence the other fundamental locus for the Botryosphaeriaceae identification, tubulin.

Of course it would be crazy to re-open the phylogenetic analysis, but at least, right because you consider it a new species, having the TUB sequences deposited in Genbank would be nice for future studies. And maybe, you can also mention and discuss something about nucleotide differences in TUB between your new species and L. citricola.

But I confirm, the analysis and phylogenetic tree are good and I consider them great to achieve the aim of this study.

**********

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Reviewer #1: Yes: Benoit LAURENT

Reviewer #3: No

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19 Mar 2020

Answers to the academic editor

I recommend starting out the introduction with a wider scope. Citrus cultivation is not only important in Algeria but in many countries and the fruits are being exported and eaten in even more countries, therefore I would start out with some sentences about the importance of Citrus in general. This could make the publication more attractive to a wider readership.

R: Revised as recommended

The authors mention at the end of the introduction that Botryosphaeriaceae have not been studied in detail in Algeria. Have other pathogen families or groups on Citrus already been studied in more detail? I think this should be introduced here in detail! This could also explain why the authors only focus on Botryosphaeriaceae and not check which other pathogens are causing the symptoms in their samples. Please explain that in more detail!

R: In this part of the introduction, we mentioned that there has not been any thorough study about citrus in Algeria. Until today, trunk diseases caused by Botryosphaeriaceae species has been widely reported in Algeria and has been associated with many important crops such as grapevine, date palm and some other fruit and forest trees. This fact led us to focus only on Botryosphaeriaceae in this study.

Regarding citrus dieback, the only previous study done so far is the one published by Linaldeddu et al. (2015) where we described Lasiodiplodia mediterranea based on few samples. Thus, in the present study, we decided to collect more samples from several orchads in order to sudy the diversity of the Botryosphaeriaceae and their association with citrus diaback. Indeed, this study represents the most comprehensive study on the presence, diversity and pathogenicity of Botryosphaeriaceae species associated with declining citrus trees in Algeria.

The objectives at the end of the introduction should be described in more detail! The authors do not even mention that they also aimed at testing the aggressiveness of the the isolates!

R: Revised as recommended

Table 1, I agree with Benoit Laurent that it would be relevant (if possible) to add the GPS coordinates of each orchard in each region as it would give an idea of how far apart the orchards are. If this information cannot be given, I would rather remove the GPS coordinates of the cities.

R: Revised as recommended (Unfortunately, we were not able to get the exact GPS coordinates of each orchard. Thus, as advised, we removed the GPS coordinates from the table).

l. 145 Mention which was the best fitting DNA evolution model.

R: Revised as recommended

Please improve the description of the statistical analyses and test specifically for differences in aggressiveness between species as suggested by Benoit Laurent.

R: Revised as recommended

I am a little surprised about the paragraph called “Nomenclature”. I am not familiar with the description of new fungal species but I have never seen such a justification. Is this typical? Otherwise, it should be removed. I suggest to name this paragraph “Taxonomy” which should be mentioned that it has been described in detail and that the new names have been submitted to MycoBank.

R: Regarding the Nomenclature, we included it because it was one of the requirements of the journal, for the description of new species. I believe that adding it or removing it all depends on the choice of the editor. I checked two papers, one of them has it and the other does not.

In the results the authors mention that they obtained 47 fungal colonies belonging to Botryosphaeriaceae. Did they also observe other fungi in their isolates? What happened to the other samples?

R: In this study, Botryosphaeriaceae species accounted for more than 50% of the isolates, making this by far the most common taxonomic group associated with branch cankers in declining citrus trees. As mentioned previously in the introduction, we wanted to focus our study only on Botryosphaeriaceae due to their important impact worldwide. Regarding the remaining isolates, in addition to the wood samples which did not develop any fungus, we obtained the following genera: Alternaria, Pestalotiopsis and some sterile fungi, which we could not identify. These samples were not considered in the study due to their low frequency as well as to our focus on Botryosphaeriaceae.

l. 41 clementines

R: Revised as recommended

l. 62-64 This sentence is unclear. Do the authors refer to a single species of the Botryosphaeriaceae family or several that are all important pathogens? Please clarify!

R: Towards the end of the same paragraph, we have mentioned all the Botryosphaeriaceae species belonging to several genera that impact the citrus, as described in different studies.

l. 77 …citrus trees in Algeria…

R: Revised as recommended

l. 350 …similar situations have been described…

R: Revised as recommended

l. 351 …according to some authors… is not a good formulation!

R: Revised as recommended (we replaced it by, ‘According to previous studies’)

Answers to the reviewer 1

General comments:

Reviewer #1: I would like to thank Akila Berraf-Tebbal and his collaborators to the answers they brought to my questions and comments. The data on the tef1 alpha are now available, which was one major concern of my last review, and allow me to verify the monophyletic group corresponding to the introduction of a new species. The debate on this introduction is still questionable but does not question the quality and the presentation of the work. I don’t think that following a trend is really constructive and yet scientific, but I endorse their point of view about the dynamicity of the taxonomy.

Besides, I’m rather surprised that, in response to my comment about the lack of general information on Botryosphaeriaceae, a sentence is brought from a reference and citing “It comprises 24 genera encompassing 222 species”, which is a rather static way of introducing the phylogeny of Botryosphaeriaceae. I would rather say “It comprises at least 24 genera encompassing 222 species”, or “It comprises at least 24 known genera encompassing 222 known species”.

R: Revised as recommended

Overall, I find the manuscript clearer as it is and have only one major suggestion to make this manuscript in the standard for publication. It concerns the pathogenicity test, the statistical analysis conducted on it and its interpretation. First of all, I suggest to add in the material and method (Pathogenicity test section) that lesion size was used as a proxy to measure the aggressiveness of the isolate. As you mentioned to one of the other reviewer comment, aggressiveness is a complex trait and proxies are often used, and are always good to define in the text.

R: Revised as recommended (we mentioned that internal lesion length was used as a proxy to measure the aggressiveness of the isolates)

Then, the "Statistical analyzes" section from the Materials and Methods has still a margin of improvement. In your case, the ANOVA was used to test the genotype effect on the lesion size variation, with significance accepted for P<0.05. The test used to test the normality of the data could be also presented. Finally, what you can say so far is that some isolates belonging to some species was more aggressive than other isolates, belonging maybe to other species. But, you didn’t test the species effect, which would appreciate the variation of the means per species and test if the means are different. Hence you cannot say that one species is more aggressive than another one (L403 in the discussion, and the whole paragraph suggest so as it is presented). If the standard deviation is too important (D. seriata for example), therefore you’ll need more genotype per species to reach statistical significance and confirm those species effects, and then used HSD to identify which species is statistically different from the others. Therefore, I suggest to adapt the discussion in consequence, or add more genotypes in the pathogenicity test if possible.

Finally, I hope this section will be more accurately presented and this manuscript will be published as the results from this work will be beneficial for the community.

R: Revised as recommended (The Shapiro-Wilk test conducted for normality checking was presented. As for the species effect, we agree with the reviewer that we need to conduct further tests on other isolates for each species in order to get clear conclusion about differences in aggressiveness between species. Unfortunately, due to some circumstances, it would be almost impossible to perform these tests. Thus, we made sure to adapt the discussion accordingly and take into consideration the reviewer’s comments.)

Minor comments:

I’m surprised by the answer of one of my comment about the basidiocarp observed in some citrus orchard:“Basidiocarps are the fruiting bodies of the decay-causing fungi. Their presence on the trunk means that it is an already rotting trunk and that some ascomycetes (Botryosphaeriaceae, Diatrypaceae…) and Basidiomycetes (Fomitiporia, Phellinus….) have already colonized the trunk.” Is that always true? For me, some decay-causing fungi are able to provoke the rotting of the trunk by themselves (e.g. Heterobasidion on pine trees).

R: I agree with the reviewer. What I meant, back in the first review, is that in our case, all the basidiocarps were observed in already rotting trunk. Indeed, it is true that it is not always the case. As the reviewer mentioned, some decay-causing fungi are able to provoke the rotting of the trunk by themselves.

L95: There may have a misunderstanding in my comment. I was more interested in the GPS location of the orchards than the GPS location of the city. If the authors don’t have access to this information, this is not a big deal. The aim of my comment was to give additional information that could feed the discussion about the distribution pattern of the Bot species. Was L. mitidjana sp. nov. isolated both year? In similar orchard (tree ages?)

R: Regarding the GPS coordinates, unfortunately, we were not able to obtain the exact coordinates of the orchards. As advised by the editor, we will remove the city coordinates since they are not relevant. Regarding the isolates, different samples from different orchards have been collected during the time period from 2013 to 2015. Thus, replying to question of the reviewer, we did not collect samples from the same orchard each year. All the prospected orchards had approximately the same age (between 25 and 30 years old).

L374: “L. mitidjana sp. nov. was found in the five surveyed orchards. “ suggests that L. mitidjana sp. nov. was isolated from all the surveyed orchards. I suggest “L. mitidjana sp. nov. was found on five orchards out of ten”, or “L. mitidjana sp. nov. was found in five of the surveyed orchards”.

R: Revised as recommended

Furthermore, do you have some elements to discuss the distribution of the species? Is there any particularity on these locations? The table containing all the information about species distribution and presented in response to my comment is particularly interesting. I’m surprised this table is not presented, at least in supplemental file.

R: Revised as recommended (the table is in the supplementary files)

L397: “our country” is maybe to personal

R: Revised as recommended (we replaced it by Algeria)

Answers to the reviewer 3

Reviewer #3: Dear Authors

I found this piece of work really interesting, well done, with a very good language, robust methodology and, much important, the article has been strongly improved after the first revisions.

A very few small notes are included in the attached pdf.

R: Revised as recommended

Furthermore, my only big concern, as mentioned by another reviewer, is about the description of the new Lasiodiplodia species. But I have read what you authors already have replied to the first reviewer and I can understand your point of view.

So, I accept your reasons and your determination to consider this as a novel species.

Probably, just to be clearer in the manuscript, it could be useful to sequence the other fundamental locus for the Botryosphaeriaceae identification, tubulin.

Of course it would be crazy to re-open the phylogenetic analysis, but at least, right because you consider it a new species, having the TUB sequences deposited in Genbank would be nice for future studies. And maybe, you can also mention and discuss something about nucleotide differences in TUB between your new species and L. citricola.

But I confirm, the analysis and phylogenetic tree are good and I consider them great to achieve the aim of this study.

R: Thank you for your comments and for understanding. We agree with your point. We could sequence the TUB and deposit it in GenBank and have them available for future studies. To be honest, in the meantime, it is hard to sequence the TUB due to the present circumstances and all what is happening around the word. We agree that this may be debatable. In fact, we have discussed this previously with the second reviewer. However, our point is that our work is clearly aligned with the current trend for introduction of novel Lasiodiplodia species. In fact, in all studies, TEF1 is considered as the most informative locus used to ID species for the Botryosphaeriaceae. We understand that the taxonomy is dynamic and hence frequently changing. Yet, for the moment we would like to introduce the new species. The fact is that eventually it will be described as a new species, if not by our group, then by someone else. This is why, if possible, we would appreciate if the acceptance for publication of this manuscript is not delayed because of that.

As an example of a case which is similar to ours, here are the nucleotide differences for the following mentioned species: L. chinensis vs L. lignicola vs L. pseudotheobromae. For all 3 species ITS is 100 % identical. We aligned the TUB sequences of these species and obtained 100% of similarity between them. As for Tef1: L. chinensis vs L. lignicola: 1 nucleotide difference L. chinensis vs L. pseudotheobromae: 3 nucleotides differences

The authors cite the articles number 8,9,10,11. I don’t think the paper number 11 is related with Diaporthe in Europe. It should be removed. On the contrary, recent studies on DIaporthe which should be cited are:

-Guarnaccia, V., & Crous, P. W. (2018). Species of Diaporthe on Camellia and Citrus in the Azores Islands. Phytopathologia Mediterranea

-Guarnaccia, V., & Crous, P. W. (2017). Emerging citrus diseases in Europe caused by species of Diaporthe. IMA fungus, 8(2), 317-334.

R: Revised as recommended (we removed the reference 11 and added the suggested ones)

Personally, I agree with this atypical inoculatin way, I mean for the cut twigs. However, it should be better if the authors support this method with other article(s) where the same method has been used on Citrus spp.

R: Revised as recommended

Please start the sentence with the entire name Lasiodiplodia. Do not abbreviate the first word.

R: In this particular line, we did not include the full name since it has already been cited using the entire name when it has been first mentioned in the manuscript.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

16 Apr 2020

Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria

PONE-D-19-29810R2

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Additional Editor Comments:

The manuscript “Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria” has received two rounds of review. The authors have satisfactorily taken into account the comments raised by the three independent reviewers and myself and I would like to congratulate the authors for the revised manuscript. Concerning the paragraph on the “Nomenclature”, I agree with the authors to include it, as it conforms to the guidelines of PLOS ONE. The methodology is clear and concise and the manuscript has improved considerably during the revision process, therefore I recommend publication. However, the following very minor comments should still be taken into account in the final version of the manuscript.

l. 152-153: Not clear! Maybe: A discrete Gamma distribution with five categories was used to model evolutionary rate differences among sites.

l. 194-195 Please insert the names of the digital repositories. All PLOS articles are deposited in PubMed Central and LOCKSS. If your institute, or those of your co-authors, has its own repository, we recommend that you also deposit the published online article there and include the name in your article.

l. 420 …contradict a previous study…

l.421 Did the authors in reference 62 compare the aggressiveness of the same species as in the present study? Please mention this here.

I did not notice before but typically, family names, such as Botryosphaeriaceae are not written in itallics, please correct.

24 Apr 2020

PONE-D-19-29810R2

Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria

Dear Dr. Berraf-Tebbal:

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