A New Leaf Blight Disease Caused by Alternaria jacinthicola on Banana in China

: A leaf blight disease with an incidence level of about 50% was found on Robusta banana in Guangdong province of China in September 2020. The early symptom appeared as pale gray to black brown, irregular, small, necrotic lesions mainly on the top 3–5 leaves. Severely infected leaves were withered and necrotic. Two representative fungus strains, strain L1 and strain L2, were isolated from affected banana leaves, and morphological and molecular identiﬁcation analysis conﬁrmed that the two fungi were both Alternaria jacinthicola . Many Alternaria species have been reported to cause wilting, decay, leaf blight and leaf spots on plants and lead to serious economic losses in their production, including A. alternata , causing leaf blight and leaf sport diseases on banana. The Koch’s postulates of A. jacinthicola causing the leaf blight disease was further fulﬁlled, which conﬁrmed that it is the causal agent of this disease. To our knowledge, this is the ﬁrst report of A. jacinthicola causing leaf blight on banana in China.


Introduction
Banana (Musa spp.), one of the most popular and widely grown fruit and a major source of food crop, is a staple for more than 400 million people and a huge commercial export [1].China is the one of the largest banana-growing and -producing countries of the world.Moreover, the banana industry has been considered as an important economic pillar for many farmers in south China.As cultivated banana cultivars are generally low in disease resistance, the development of the banana industry has been greatly constrained by many infectious diseases caused by pathogenic microorganisms.Among these diseases, banana wilt disease and several leaf diseases have caused great yield and economic losses [2,3].The banana wilt disease (also called Panama disease or Fusarium wilt disease) caused by the soil-borne fungus Fusarium oxysporum f. sp.cubense (Foc) was uncontrollable yet and is, thus, recognized as the most destructive one.Therefore, its control has received great attention and substantial funding.The banana leaf diseases, however, were much less focused compared to the former disease.Leaf diseases can be suppressed or controlled by applying chemical or biocontrol agents, by strengthening field management and agrotechnical control, and by using integrated control methods.However, what we cannot ignore is that the prevention and control cost of leaf diseases accounts for more than 20% of the total disease control cost of banana [4][5][6][7].Leaf diseases directly affect the yield and quality of bananas, and can also lead to premature ripening of bananas [8,9].Many fungi have been identified as pathogenic fungi causing severe leaf diseases on banana [10][11][12][13].
Alternaria is a widespread fungus genus including more than 250 species, and it can be found in a variety of plant species [14].Accumulated evidences have shown that a warm and moist environment is favorable for the spread and infection of Alternaria fungi [15].After infection, the fungus will produce toxins that damage plant tissues [14].Some Alternaria species have been identified to be causal pathogens of kiwifruit leaf spot [16], citrus brown spot [17], apple leaf blotch and fruit spot [18], and tobacco brown spot disease [19].Moreover, several Alternaria species have also been identified as causal agents of banana leaf spot disease [20][21][22][23].Among these Alternaria species, A. jacinthicola has become an increasing important pathogen causing leaf diseases to a broad host range.The fungus can cause leaf spot and leaf blight diseases, which has been reported in water hyacinth and marigold [24,25].A. jacinthicola is not easily distinguishable from other Alternaria species from morphological level.And molecular classification analysis also shows that A. jacinthicola has only a few specifically conserved nucleotides.What is worse, most of the nucleotide differences present between A. jacinthicola and the A. alternata isolates are also present in the A. tomato and/or A. burnsii isolates [26].Therefore, it is very necessary to distinguish it from other Alternaria species when determining the causal agent of some certain diseases.
During November 2020, a leaf disease on Robusta banana (Musa acuminata cv.Giant Cavendish, AAA Group) with an incidence level of about 50% was observed in the banana germplasm evaluation orchard of the Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences located in Suixi county, Zhanjiang city, Guangdong Province.The disease symptoms were somewhat similar to the reported leaf blight diseases caused by Alternaria species [21][22][23].The aim of the study was to identify the causal agent of this disease based on morphological and molecular characteristics.

Results
The leaf disease on Robusta banana (Musa acuminata cv.Giant Cavendish, AAA Group) with an incidence level of 50.68% was observed in the banana germplasm evaluation orchard of Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences located in Suixi county, Zhanjiang city, Guangdong Province.Mildly diseased leaves showed pale gray to black brown, irregular, small, necrotic lesions, and these symptoms were mainly found on the top 3-5 leaves.Severely infected leaves were withered and necrotic (Figure 1A,B).To isolate the causal pathogen, 10 diseased leaves were collected and used for fungi isolation and purification.We obtained two fungus strains, strain L1 and strain L2, frequently isolated form diseased leaves.The colony characteristics and conidial morphology these two fungus strains were further observed and analyzed.Their colony mycelia were white to dark brown that gradually changes to black on the underside on PDA medium (Figure 1C,D).Its mycelium was septate and its conidia were variable in size and shape (mostly obclavate and tapering in the lower half).The conidia were usually in chains, as well as solitary, ellipsoid or obclavate, and smooth walled, while the upper part of the conidium bears a short and rounded beak.Conidia were measured to be 13 to 56 × 8 to 15 µm in size (n = 50) with two to nine transverse septa and one to three longitudinal septa.The beak was 4 to 20 × 2 to 4 µm in size (n = 50), with 0 or 1 septa (Figure 1E,F).The morphology resembled descriptions of the small-spored A. jacinthicola, A. burnsii, and A. tomato [27,28].Thus, the fungus strain L1 and L2 were primarily identified as Alternaria sp.[26].
To further determine the fungal pathogen causing leaf blight on Robusta banana, four phylogenetic trees were constructed using GAPDH, TEF, Alt a1, and tandemly linked ITS-GAPDH-TEF-Alt a1 sequences of strain L1 and strain L2 and corresponding sequences from some other Alternaria species, respectively.A phylogenetic tree constructed based on GAPDH sequences showed that the relationship between the two strains and A. jacinthicola isolate strain F217169 was the closest (Figure 2A).A phylogenetic tree constructed based on the TEF gene sequences showed that Strain L1 was closest to A. jacinthicola isolate strain F217169, but strain L2 was close to A. jacinthicola, A. alteranta and A. burnsii (strain F217169, 170242, and NCYU 19-0144) (Figure 2B).A phylogenetic tree constructed based To further determine the fungal pathogen causing leaf blight on Robusta banana, four phylogenetic trees were constructed using GAPDH, TEF, Alt a1, and tandemly linked ITS-GAPDH-TEF-Alt a1 sequences of strain L1 and strain L2 and corresponding sequences from some other Alternaria species, respectively.A phylogenetic tree constructed based on GAPDH sequences showed that the relationship between the two strains and A. jacinthicola isolate strain F217169 was the closest (Figure 2A).A phylogenetic tree constructed based on the TEF gene sequences showed that Strain L1 was closest to A. jacinthicola isolate strain F217169, but strain L2 was close to A. jacinthicola, A. alteranta and A. burnsii (strain F217169, 170242, and NCYU 19-0144) (Figure 2B).A phylogenetic tree constructed based on Alt a1 gene sequences showed that both strain L1 and L2 were closest to A. jacinthicola (YZU 171616, COUFAL0255) (Figure 2C).Moreover, the phylogenetic tree constructed based on the tandemly linked ITS-GAPDH-TEF-Alt a1 sequences showed that strain L1 and strain L2 were both closest to A. jacinthicola (Figure 2D).Thus, this causal fungus of the leaf blight disease on Robusta banana was identified as A. jacinthicola.
the leaf blight disease on Robusta banana was identified as A. jacinthicola.
To prove pathogenicity, Koch's postulates were verified on detached banana leaves according to the method described by Li et al. [33].At 4 days post inoculation, typical leaf blight symptoms appeared on all the fungi inoculated banana leaf wounds, while control wounds remained symptomless (Figure 1G).The pathogen was successfully and consistently re-isolated from diseased lesions.After molecular identification of the re-isolated fungus based on ITS, TEF, GAPDH, and Alt a1 genes sequencing results, the Koch's postulates of A. jacinthicola were fulfilled.

Discussion
Alternaria is a fungus genus with a wide host range, causing wilting, decay, leaf blight, and leaf spot of many crops and fruits.Pragya et al. [21] reported that A. tomatophila and A. alternata were able to cause early blight disease in tomato, and their diseased areas were characterized by dark, small, necrotic, coalescing, and concentric lesions on the leaf surface.Rodrigues et al. [34] demonstrated that A. tomatophila was able to infect not only tomato but also potato plants, experimental inoculation tests showed brownish lesions on To prove pathogenicity, Koch's postulates were verified on detached banana leaves according to the method described by Li et al. [33].At 4 days post inoculation, typical leaf blight symptoms appeared on all the fungi inoculated banana leaf wounds, while control wounds remained symptomless (Figure 1G).The pathogen was successfully and consistently re-isolated from diseased lesions.After molecular identification of the reisolated fungus based on ITS, TEF, GAPDH, and Alt a1 genes sequencing results, the Koch's postulates of A. jacinthicola were fulfilled.

Discussion
Alternaria is a fungus genus with a wide host range, causing wilting, decay, leaf blight, and leaf spot of many crops and fruits.Pragya et al. [21] reported that A. tomatophila and A. alternata were able to cause early blight disease in tomato, and their diseased areas were characterized by dark, small, necrotic, coalescing, and concentric lesions on the leaf surface.Rodrigues et al. [34] demonstrated that A. tomatophila was able to infect not only tomato but also potato plants, experimental inoculation tests showed brownish lesions on inoculated potato leaves.A. alternata has been identified to be pathogen causing citrus brown spot and watermelon leaf blight [35,36].Its diseased symptoms on citrus are brown to black lesions surrounded by a yellow halo, and leaf drop if severely infected [35].In watermelon, A. alternata induced dark brown leaf spot lesions similar to the symptoms that appeared in citrus [36].Moreover, it was reported that Alternaria species can infect more than one host plant [37].Consistently, A. alternata has been previously described as pathogenic fungus on banana, causing symptoms of light to dark brown spots and eventually leaves wither and fall.Parkunan et al. [38] reported that A. alternata infection could cause leaf spot symptoms in seven banana cultivars including Novaria, Cavendish, and so on, causing light to dark brown spots on the adaxial surface of the leaves and occurred on all except the newly emerged leaves.In 2014, Fu et al. [39] also found that A. alternata caused similar disease symptoms, irregular spots with gray to off-white centers surrounded by dark brown margins, and usually surrounded by chlorotic halos mainly on the old leaves of Chinese dwarf banana (Ensete lasiocarpum).
In this study, we reported a leaf disease on Robusta banana in China.According to the symptoms in diseased leaves, it was defined as a banana leaf blight disease.Pathogen isolation and morphological observation results showed that the disease was caused by Alternaria species.It is not easy to distinguish Alternaria species by their morphology differences [24].Our morphology identification results showed that the potential pathogens we isolated from diseased banana leaves were more likely to be A. jacinthicola or A. burnsii or A. tomato.BLASTn results showed that the ITS sequences of isolates L1 and L2 were 100% identical to those of other Alternaria species, making it unable to determine the species of the causal pathogen.Woudenberg et al. [26] pointed out that Alternaria species could be distinguished using several gene sequences including GAPDH, Alt a1, and so on.By further utilizing TEF, GAPDH, and Alt a1 gene sequences, the causal pathogen was determined.Phylogenetic analysis results showed that the causal pathogen was A. jacinthicola.A. jacinthicola has been reported as a plant pathogen causing blight leaf disease in water hyacinth [24] and leaf spot disease in Tagetes erecta [25].To the best of our knowledge, this study represents the first report of banana leaf blight caused by A. jacinthicola in China.

Disease Investigation and Sample Collection
On 20 November 2020, symptoms of diseased Robusta banana were firstly observed and recorded in the banana germplasm evaluation orchard of Institute of Fruit Tree Research, Guangdong Academy of Agricultural Sciences located in Suixi county, Zhanjiang city, Guangdong Province, China.Disease incidence level was calculated using the formula: incidence lever (%) = diseased Robusta banana seedling number/Robusta banana seedling number in the orchard ×100.Then, diseased leaf samples were harvested and brought back to the lab on ice for pathogen isolation.

Isolation and Purification of Pathogenic Fungi
Causal pathogen isolation experiment was performed according to the method described by Kant et al. [22] with modifications.Briefly, 5 mm × 5 mm leaf sections were excised from lesion margins, surface-disinfected with 75% ethanol for 90 s, rinsed with sterile water for 30 s for three times, cultured on potato dextrose agar (PDA, containing 200 g/L potato, 20 g/L glucose and 20 g/L agar) plate and incubated at 28 • C in dark for 2 d.Hyphae grown from the diseased sections were transferred to fresh PDA plate for two to three rounds of purification.

Morphological Observation and Molecular Identification of Pathogenic Fungi
Two representative potential fungal species, strain L1 and L2, were consistently isolated from diseased banana leaves.After culturing them on PDA at 28 • C for 7 d, the morphology and coloration of the colony were observed and recorded under a microscope (Leica DMIL).
The mycelium was washed off from the PDA medium using distilled water, collected and frozen with liquid nitrogen, and grounded into fine powders for DNA isolation.Total genomic DNA was extracted by using Fungus Genomic DNA Extraction Kit (Bioer Technology, Hangzhou).PCR reactions were performed using the universal fungus inter-nal transcribed spacer (ITS) primer pair ITS1/ITS4 [29], partial translation elongation factor 1-α (TEF) primer pair EF1-728F/EF1-986R [30], glyceraldehyde-3-phosphate dehydrogenase (GAPDH) universal primer pair gpd1/gpd2 [31], and Alternaria major allergen (Alt a1) specific primer pair Alt F/Alt R [32].The 25 µL PCR mixture contained 12.5 µL of Dream Taq TM Green PCR Master Mix (2×) DNA polymerase, 1 µL of each primer, 9.5 µL of double-distilled water, and 1 µL of DNA template.The PCR condition was set as follows: pre-denaturation at 95 • C for 3 min; denaturation at 95 • C for 30 s, annealing at 52 • C for 30 s, elongation at 72 • C for 30 s, 38 cycles; and a final extension at 72 • C for 10 min.PCR products were detected in 1% agarose gel and were then subjected to Sanger sequencing by the Biosune Biotechnology Company, Shanghai, China.ClustalW embedded in MEGAX was used for multiple sequence alignment of ITS, TEF, GAPDH, and Alt a1 sequences from Alternaria species.Phylogenetic trees were constructed using Maximum Likelihood (ML) method with 1000 repetitions, and other parameters were set as default.

Pathogenicity Identification
To prove pathogenicity, Koch's postulates were verified on detached banana leaves according to the method described by Li et al. [33].Briefly, healthy leaves detached from five-leaf banana seedlings were placed in sterile Petri dishes (140 mm) on sterile filter papers.Leaves were wounded with a sterile needle to make three wounds for each side of the leaf.Then mycelial plugs were placed over the wounds on one side, and agar plugs were placed over the other side as controls.To maintain high relative humidity, 15 mL sterile H 2 O was added to the sterile filter papers.Experiments were conducted with eight replications.After symptom appearance, pathogens were again isolated, purified, and molecularly identified using ITS, TEF, GAPDH, and Alt a1 primer pairs [34,35].

Conclusions
In our present study, through morphological and molecular identification, A. jacinthicola was identified as the pathogen causing leaf blight disease on Robusta banana (Musa acuminata cv.Giant Cavendish, AAA Group).Given that the leaf blight symptoms produced by different pathogens were similar and confusing, the molecular pathogen identification result obtained in this study will be helpful for the future control of this disease.

Horticulturae 2021, 7 , 8 Figure 1 .
Figure 1.Leaf blight disease symptoms on banana and morphology of the causal pathogen.(A) Leaf blight disease symptoms in the field; (B) Leaf blight-diseased leaves; (C,D) Colonies of Alternaria jacinthicola on PDA (C, reverse; D, obverse); (E,F) Representative hyphae and conidia of A. jacinthicola; (G) Leaf symptoms at 4 days after A. jacinthicola inoculation; the left leaf part was inoculated with A. jacinthicola agar discs and the other part was inoculated with PDA agar discs.

Figure 1 .
Figure 1.Leaf blight disease symptoms on banana and morphology of the causal pathogen.(A) Leaf blight disease symptoms in the field; (B) Leaf blight-diseased leaves; (C,D) Colonies of Alternaria jacinthicola on PDA (C, reverse; D, obverse); (E,F) Representative hyphae and conidia of A. jacinthicola; (G) Leaf symptoms at 4 days after A. jacinthicola inoculation; the left leaf part was inoculated with A. jacinthicola agar discs and the other part was inoculated with PDA agar discs.

Figure 2 .
Figure 2. Phylogenetic trees constructed using GAPDH, TEF, Alt a1, and tandemly linked ITS, GAPDH, TEF, and Alt a1 sequences from Alternaria species.(A) Phylogenetic tree constructed using GAPDH gene sequences from different Alternaria species.(B) Phylogenetic tree constructed using TEF gene sequences from different Alternaria species.(C) Phylogenetic tree constructed using Alt a1 gene sequences from different Alternaria species.(D) Phylogenetic tree constructed using tandemly linked ITS, GAPDH, TEF, and Alt a1 sequences from different Alternaria species.The numbers at the nodes indicate the bootstrap values calculated for 1000 repetitions.The isolated strain L1 and strain L2 is shown in bold and triangle.

Figure 2 .
Figure 2. Phylogenetic trees constructed using GAPDH, TEF, Alt a1, and tandemly linked ITS, GAPDH, TEF, and Alt a1 sequences from Alternaria species.(A) Phylogenetic tree constructed using GAPDH gene sequences from different Alternaria species.(B) Phylogenetic tree constructed using TEF gene sequences from different Alternaria species.(C) Phylogenetic tree constructed using Alt a1 gene sequences from different Alternaria species.(D) Phylogenetic tree constructed using tandemly linked ITS, GAPDH, TEF, and Alt a1 sequences from different Alternaria species.The numbers at the nodes indicate the bootstrap values calculated for 1000 repetitions.The isolated strain L1 and strain L2 is shown in bold and triangle.