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.

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' Unfunded studies Enter: The author(s) received no specific funding for this work.  Sequences of all Lasiodiplodia species known from culture were retrieved from GenBank 138 (S1 Table) and aligned with sequences of the isolates obtained in this study. Alignments were containing sterilized water as a growth substrate. They were incubated at the ambient room 164 temperature, under daily photoperiod. The water of the container was changed twice a week.

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There were 10 replicates per isolate, and the same number of cuttings was used as controls.    showed significant difference in lesion length between its two isolates (Table 3).

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At least, two different species were found in each orchard. L. mediterranea and Doth. 338 viticola were found in six of the ten surveyed orchards. They were followed by L. mitidjana, 339 recorded from five orchards of two municipalities. D. seriata was found in four sampling 340 sites; whereas, D. mutila was recovered from only two orchards of the same municipality. Algeria. The Botryosphaeriaceae species were recovered from more than half of the trees 359 sampled and were found in all the prospected orchards.

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Lasiodiplodia was the most commonly isolated genus that was found in six of the ten 361 surveyed orchards. This fact is consistent with previous studies, which showed that  Isolates of this species were present predominantly in the wedge-shaped necrosis.                                           citrus trees has not been studied in detail. Therefore, the aim of this study was to investigate 77 3 and determine the incidence of the Botryosphaeriaceae species associated with branch canker 78 and dieback in the major citrus-growing region of Algeria.

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No specific permits were required for the described field studies. This study did not involve 82 endangered or protected species.

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Field survey and sampling 84 Surveys were conducted in ten commercial orchards in the northern region of Algeria.    Sequences of all Lasiodiplodia species known from culture were retrieved from GenBank 138 (S1 Table) and aligned with sequences of the isolates obtained in this study. Alignments were  (Table S1), forming a single monophyletic group. The second group contained 14 236 isolates, which formed a distinct clade, with a high bootstrap support (ML/MP = 80/94), was 237 considered to represent a distinct species, which is described here as Lasiodiplodia mitidjana 238 sp. nov. (Fig 2).   3. Lasiodiplodia mitidjana. (a,b). Pycnidia formed on pine needles. (c).  showed significant difference in lesion length between its two isolates (Table 3).

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At least, two different species were found in each orchard. L. mediterranea and Doth. 338 viticola were found in six of the ten surveyed orchards. They were followed by L. mitidjana, 339 recorded from five orchards of two municipalities. D. seriata was found in four sampling 340 sites; whereas, D. mutila was recovered from only two orchards of the same municipality. All the Botryosphaeriaceae species of this study caused necrosis on the citrus shoots, with 402 differences in the lengths of the lesions. These differences were observed between the species 403 and also among isolates of the same species. Thus, the results suggest that Diplodia seriata 404 and L. mediterranea could be considered as being the most aggressive, since they produced 405 the longest lesion. However, D. seriata was significantly different compared to the rest of the 406 isolates, which is consistent with previous studies that showed significant impact of D.