Diversity among Isolates of the Tangerine Pathotype of Alternaria alternata Andréa

The tangerine pathotype of Alternaria alternata is the aetiological agent of Alternaria brown spot on tangerines. In the state of Paraíba, Brazil, its occurrence on “Dancy” tangerine trees is associated with genetic aspects as well as the influence of environmental conditions on reproduction and dissemination within and between populations. The aim of the present study was to evaluate the diversity of isolates of this pathogen using morphophysiological and molecular markers. For the analysis of mycelial growth and sporulation, 30 isolates from different locations were examined at 24-hour intervals until the seventh day, when the spores were quantified. The 30 isolates were characterised based on molecular markers (ISSR) and genetic similarity (Jaccard index). A factor arrangement was used: 30 isolates, four media (ODA, PDA, LEA and V8), three light regimes (continuous dark, alternating light and continuous light) and three temperatures (15 ̊C, 25 ̊C and 35 ̊C), with 12 repetitions. Groups 1, 2 and 3 presented low genetic variability. Group 4 showed high genetic variability of the isolates obtained from the Massaranduba (state of Paraíba-Brazil) producing region and higher mycelial growth and sporulation of A. alternata. The continuous light regime and the temperature 25 ̊C in PDA and V8 media were the ideal conditions for the mycelial growth and sporulation, respectively, of the isolates of A. alternata.


Introduction
Alternaria diseases of citrus caused by Alternaria spp.(mainly Alternaria alternata) are important diseases with worldwide distributions.They have been do- cumented in the USA [1], Turkey [2], Spain [3], Argentina, Brazil [4], China [5] and Iran [6].Different diseases caused by Alternaria spp. on citrus species include brown spot of tangerines (Citrus reticulata Blanco) and their hybrids (namely the tangerine pathotype), leaf spot of rough lemon (Citrus jambhiri Lush) (namely the rough lemon pathotype), black rot of fruits and leaf spot on Mexican lime [7].
Alternaria brown spot, caused by the fungus Alternaria alternata (Fr.:Fr.)Keissler is the principal fungal disease of tangerines (Citrus reticulate) and their hybrids.This disease is considered a biggest problem on the fruit of many citrus cultivars grown for fresh markets.It causes lesions on leaves, stems, and fruit and reduces yield and fruit quality [5].The symptoms of Alternaria brown spot are small black or brown spots on young leaves and small lesions with a corky appearance, with or without a chlorotic halo, which occur on the twigs and fruits [8].
This fungus produces a toxin that is responsible for most if not all of the disease symptoms.Sometimes the toxin is translocated up the veins and produces a necrosis of the veinlets that is characteristic of the disease.On highly susceptible cultivars, entire areas of the leaf may be killed and the leaves may abscise [9].
Most spores of Alternaria are produced by lesions on mature leaves on the tree, or recently fallen infected leaves on the grove floor.Even though spores are airborne, plantings of healthy trees will remain disease-free for long periods.If present from the outset, it builds to high populations during the period of vegetative growth on young trees and subsequently is on fruits [10].
There are more than eleven new species of Alternaria described as causal agents of the brown spot in different parts of the world, however, due to the conflicting morphological data, molecular studies have been expanded through polymorphism sequencing and mitochondrial restriction fragment length DNA and regions internally transcribed in the ribosome, where it has favored the identification of the close phylogenetic relationship between the isolates producing toxins and ensuring the establishment of identity as well as its variability [11].
The genetic variability of citrus pathotypes of Alternaria alternata is associated with the conditions of the in vivo or in vitro environment in which these fungi develop.Indeed, environmental conditions are a major cause of genetic variation within a population and exert an influence on the emergence of new genotypes and new combinations involving the fusion of gametes, the recombination of alleles during the pairing of homologous chromosomes, segregation and the generation of viable descendants [11].
In the state of Paraíba (Northeast Brazil), the occurrence of alternaria brown spot on Dancy tangerines (Citrus tangerina hort.ex Tanaka) caused by the tan-American Journal of Plant Sciences gerine pathotype of A. alternata is associated with environment factors, such as humidity, luminosity and high temperatures, which range from 25˚C to 30˚C.These conditions are important to the reproduction and dissemination of the pathogen, which causes serious harm to citrus crops, reducing productivity and the quality of fruit [12].However, little is known regarding the influence of variations in these environmental aspects on the genetic variability of A. alternata.
Thus, there is a need for studies involving methodologies in controlled environments for subsequent use of the information acquired to improve the management of Alternaria brown spot on tangerines in the field.
The effectiveness of disease control strategies depends on the understanding of the pathogen and its population dynamics [13].In this context, the study of variability in fungal populations is an important research tool.From an evolutionary point of view, the genetic variability of a population is important in determining the potential for adaptation of an organism to different environmental conditions.Furthermore, from the epidemiological point of view, pathogenic variability has direct implications for disease management [14].Therefore, it is of great importance to obtain information on the molecular analyzes of use in combination with morphological studies, which opens new avenues to find evidence of host specificity for this pathogen in the state of Paraíba, Brazil.The aim of the present study was to evaluate the diversity of isolates of the tangerine pathotype of A. alternata using morphophysiological and molecular markers.

Material and Methods
The morphophysiological analyses were performed at the Phytopathology La- Montes, Portugal, for to comparation with Brazilian isolates (Table 1).
Isolates from the state of Paraíba were obtained from leaves fragments measuring 5 mm in diameter disinfected by rinsing with 70% alcohol, 1% hypochlorite and sterilised distilled water (SDW) inoculated in potato dextrose agar (PDA).For the confirmation of morphological identity, three discs with fungal colonies measuring 5 mm in diameter were transferred to calcium carbonate  PDA, leaf extract agar [LEA-400 g of citric leaves, 20 g of dextrose, 20 g of agar and 1000 mL of SDW] and V8 agar [100 mL of V8 juice, 20 g of agar, 15 g of CaCO 3 and 900 mL of SDW]) [16].The pH of all media was adjusted to 6.0 and sterilisation was then performed in an autoclave at 120˚C for 20 minutes.
Means of mycelial growth of the treatments were performed at 24 hour intervals, with a graduated ruler the two orthogonal axes were measured until reaching the entire 13 mm diameter Petri dish and the results were expressed in (mm) until the day of evaluation [17].The sporulation averages of the treatments were in Neubauer chamber, where the suspension was adjusted to 10 5 and the results were expressed as (n˚ × 10 5 spores/mL) [18].
Among the 38 monosporic isolates used for the molecular characterisation, five fragments of mycelial discs measuring 5 mm 2 were transferred to plastic Falcon tubes.Each tube contained 30 mL of PDA and was agitated for two hours twice a day for seven days at room temperature (25˚C ± 2˚C).After the mycelial growth of the isolates, the PDA broth was removed with a vacuum pump and the mycelial mass of each isolate was filtered, pulverised in liquid nitrogen and placed into 2 mL microtubes.To obtain inter simple sequence repeats (ISSR), three primers were used that produce greater polymorphism in the bands of the electrophoresis gels with the polymerase chain reaction (PCR) of the isolates analysed [19].DNA extraction was performed in a microcentrifuge with the cetyltrimethylammonium bromide method [20] adapted for the UltraClean® Microbial DNA extraction kit using the protocol described by the manufacturer (MO BIO Laboratories Inc., Carlsbad, CA, USA).Total extracted DNA was analysed in 1% agarose gel, using GelRed Biotium® for viewing under ultraviolet stream Health, Inc., USA).Analysis of the ISSR products from the isolates was performed using the unweighted pair group method with arithmetic mean (UPGMA).The Jaccard similarity index [21] was used with a tolerance of 2% to determine the genetic similarity between the groups formed and plotted on the dendogram.
The genetic differentiation among populations was analyzed through the analysis of the genetic diversity in populations subdivided from Nei [22] [23] [24], which allows the comparing the levels of heterozygosity between and within populations, as well as obtaining an estimate of the divergence.
An entirely randomised factorial design was used for the morphophysiological characterisation, with 30 isolates, four culture media (ODA, PDA, LEA and V8),

Results
With regard to collection site, all isolates of A. alternata demonstrated differences in terms of mycelial growth and sporulation.continuous dark, alternating light and continuous light at a temperature of 25˚C in the PDA and V8 media through to the seventh day of evaluation (Table 3).
Isolates 15 to 26 demonstrated greater mycelial growth (10.68 to 10.90 mm) when incubated with continuous light at 25˚C in the PDA and V8 media.3).
Alternating light regimes, temperatures of 15˚C and 35˚C were observed, and ODA and LEA media did not influence the mycelial growth of most isolates of A. alternata until the seventh day of evaluation (Table 3).
Isolates 1, 2, 3, 4, 5 and 6 exhibited greater sporulation (3.09 × 10 5 spores/mL) when incubated in a continuous dark regime at 25˚C in the V8 medium.The same regime at 35˚C in LEA medium yielded 2.30 × 10 5 spores/mL and a continuous light regime at 25˚C in the LEA and V8 media yielded 3.60 and 1.65 × 10 5 spores/mL, respectively.These culture conditions favoured greater sporulation of the pathogen over the other culture conditions through to the seventh day of evaluation (Table 4).
The sporulation of isolates 7 to 14 was favoured when incubated in continuous dark at 25˚C in the V8 medium (1.69 × 10 5 spores/mL).At 35˚C, these isolates yielded 2.55 and 2.15 × 10 5 spores/mL in the LEA and V8, respectively.
The continuous light regime in the V8 medium stimulated these isolates to produce 1.78 and 1.65 × 10 5 spores/mL at 25˚C and 35˚C, respectively (Table 4).
The sporulation of isolates 15 to 26 was favoured when incubated in continuous dark at 35˚C in the LEA and V8 media (1.23 and 2.75 × 10 5 spores/mL, respectively).Continuous light at 25˚C in the LEA and V8 media stimulated the production of 3.83 and 2.19 × 10 5 spores/mL, respectively (Table 4).
Isolates 27, 28 and 29 exhibited greater sporulation (2.76 × 10 5 spores/mL) when incubated in continuous dark at 25˚C in the V8 medium.At 35˚C, the LEA and V8 media stimulated the production of 1.56 and 1.42 × 10 5 spores/mL, respectively.Continuous light in the V8 medium stimulated the production of 2.21 and 2.22 × 10 5 spores/mL at 25 and 35˚C, respectively (Table 4).
In continuous dark, isolate 30 produced 3.90 × 10 5 spores/mL when incubated at 25˚C in the V8 medium and 5.35 × 10 5 spores/mL at 35˚C in the LEA medium.Continuous light at 25˚C in the V8 medium stimulated the production of 3.50 × 10 5 spores/mL.These culture conditions favoured greater sporulation of the isolate of the tangerine pathotype of A. alternata in comparison to the other conditions through to the seventh day of evaluation (Table 4).
It was verified that alternating light regime, temperature of 15˚C, means ODA and PDA did not influence the production spores of most isolates of A. alternata until the seventh day of evaluation (Table 4).
Genetic diversity among the isolates demonstrated by the ISSR markers evaluated.Based on approximately 20 polymorphic bands, the isolates formed three groups with genetic similarity greater than 24% and one with 14.1%.

Discussion
There is no consensus in the literature regarding the effect of light on the biochemical mechanisms the formation of mycelial growth and conidiophores of Alternaria spp.However, the light regime exerts an important influence on sporulation in different species of phtyopathogenic fungi, such as Alternaria brassicae (Berk.)Sacc., A. solani and A. alternata [26].In the present study, continuous dark and continuous light stimulated greater sporulation of the isolates of the A. alternata.Thus, such culture conditions could be considered ideal for the greater production of spores by this pathogen.
The production of spores of A. alternata is high in prolonged periods of dark, which is in agreement with the present findings.Aragaki [27] states that the terminal phase of the sporulation cycle of different fungal species may occur in continuous darkness, since the nucleic acids and sporogenic substances that compose the conidia remain for long periods in the absence of light.Thus, sporulation is induced in filamentous fungi.A long period of light can also exert an influence on the activation of key enzymes [28] responsible for the synthesis of compounds in the culture media that are essential to the sporulation of different fungal species [29].
Based on the present study and inferring what occurs under different environmental conditions, isolates of the pathogen in agricultural crops may sporulate less than in culture media, since tangerines are submitted to an alternating Physical agents are capable of either inducing or inhibiting the vegetative and reproductive development of the majority of fungi.Temperature is one of the most important factors, as it modulates the production of proteins and enzymes involved in the maintenance of fungal cells [30] and exerts an influence on cell multiplication.Very low temperatures generally do not stimulate production in the mycelium or sporulation due to the reduction in the metabolism of the pa-thogen and very high temperatures lead to a reduction in both mycelial growth and sporulation, as some essential enzymes may denature or have their formation altered, resulting in a failure to resume metabolic activity, which can drastically reduce sporulation [12].
According to Meng et al. [31] each species of fungus has an ideal temperature range for sporulation, which is reduced at low temperatures and increases with the increase in temperature until reaching a point of maximum sporulation.Thus, a temperature range of 25˚C to 35˚C is favourable to the high production of spores in different species of Alternaria.
The highest mycelial growth of the pathotype tangerine A. alternata occurred when using potato dextrose agar, since this culture medium is rich in sources of nitrogen, phosphorus, minerals and high sugar content [32] and influence in the increase both mycelial growth.According to Pulz and Massola Júnior [16], this phase requires high concentrations of sugars, which influence the increase of vegetative development in different species of Alternaria.
The V8 agar medium exerted an influence on the greater production of spores of A. alternata.This medium is composed of eight vegetables (tomato, beet, celery, carrot, lettuce, spinach, parsley and watercress) and is rich in potassium, complex carbohydrates, minerals, fibre and vitamins A, E and C, with only 0.3% sugar, which are essential constituents for the obtainment of high sporulation in different fungal species [29].According to Brunelli et al. [30] for sporulation to occur in different species of Alternaria, the medium needs to have low sugar content [32].
The culture medium prepared with citrus leaf extract also provided significant yields of spores, since the leaves contain substances such as calcium, magnesium, zinc, phosphorus and potassium, which are essential to the reproductive phase of pathotype tangerine of A. alternata.This finding is in agreement with data described by Satyanarayana and Sadasiva Reddy [33] who report that culture media prepared with plants species that are susceptible to particular pathogens can increase the sporulation rate.Using in vitro culture studies, Leão et al. [34] found that a watermelon leaf extract medium in a continuous light regime favoured the greater production of spores by Ascochyta cucumis in comparison to the other media tested.
The physiological studies performed by Choudhary et al. [35] found that the temperature of 25˚C, PDA medium provided the best mycelial growth and sporulation of Alternaria alternata under in vitro conditions.Pulz and Massola Júnior [16] analysed the effect of 15˚C, 20˚C, 25˚C, 30˚C and 35˚C with continuous light and continuous dark in the V8, ODA and PDA media on the growth and sporulation of Alternaria dauci and found greater growth among the isolates analysed when incubated in continuous dark at 20˚C in the ODA medium, whereas a temperature of a 25˚C led to greater spore production in the V8 medium.
For Alternaria solani (Sorauer), continuous dark at 25˚C in the V8 medium were the conditions that most favoured mycelial growth and sporulation in comparison to the other culture conditions evaluated [16].
valent, such as parasexuality and parameiosis, which may have exerted an influence on the high genetic variability detected in the present study.Likewise, Michereff et al. [42] used RAPD markers in isolates of A. brassicicola and found high genetic variability as well as variability with regard to mycelial growth and sporulation.
Using ISSR, Meng et al. [31] found a diversity of genotypes of A. solani in regions in China.The authors found that 268 isolates demonstrated high genetic variation within populations of the same location and found no genetic differences between collection sites.Evaluating genetic diversity among 45 isolates of A. alternata associated with citrus trees in southern Iran using RAPD-PCR, Ghasemloee and Niazmand [38] found high genetic diversity in only 13 isolates, with no genetic divergences found among the other 32 isolates.

Conclusion
Groups 1, 2 and 3 presented low genetic variability.Group 4 showed high genetic variability of the isolates obtained from the Massaranduba (state of Paraíba, Brazil) producing region and higher mycelial growth and sporulation of A. alternata.The continuous light regime and the temperature 25˚C in PDA and V8 media were the ideal conditions for the mycelial growth and sporulation, respectively, of the isolates of A. alternata.
boratory and the molecular analyses were performed in the molecular biology sector of the Animal Product Analysis Laboratory, both of which are located in the Centro de Ciências Agrárias of the Universidade Federal da Paraíba, Campus II, Areia, Brazil.Thirty-four isolates of the tangerine pathotype of A. alternata were collected from citrus production areas in the state of Paraíba.Three isolates were obtained from the collection of the Phytopathology Laboratory of the School of Agrarian and Veterinary Sciences of the Universidade do Estado de São Paulo, Jaboticabal, Brazil, which had been obtained from symptomatic leaves of the "Fortune" tangor (C.clementina × C. tangerina) in the citrus production region of Pratânia, state of São Paulo, Brazil.One isolate of the tangerine pathotype of A. alternata was obtained from Portugal from tangerines (C.tangerina) with typical symptoms of alternaria brown spot in the region of Trás os 3 medium and transferred to test tubes containing 9 mL of SDW.Serial dilutions were performed by transferring 1 mL of suspension with 1 × 10 5 spores/mL into the third tube, distributed in three Petri dishes containing 20 g of agar and 1000 mL of SDW and incubated at room temperature (25˚C ± 2˚C) for a maximum of 12 hours or until the germination of the spores.Monosporic cultures were obtained by collecting a single spore with the aid of a light microscope and a bronze rod specifically developed for this purpose.The monosporic isolates were transferred to test tubes containing PDA and incubated for seven days at room temperature (25˚C ± 2˚C) until the time of use.The mycelial diameter of thirty isolates of the tangerine pathotype of A. alternata was measured after cultivation of the fungus under biochemical oxygen demand conditions adjusted to different light regimes (continuous dark, alternating light [12 hours of light and 12 hours of dark] and continuous light) with fluorescent light of 40 watts and lamps distant 30 cm of Petri dishes, temperatures (15˚C, 25˚C and 35˚C) and culture media (oatmeal dextrose agar [ODA-65 g of oatmeal, 20 g of dextrose, 20 g of agar and 1000 mL of SDW],

A
. C. F. Demartelaere et al.DOI: 10.4236/ajps.2018.92017200 American Journal of Plant Sciences made from spore counts (obtained by the addition of 10 mL of sterile distilled water in Petri dishes with the fungal colony, where the spores were released with the aid of a sterilized spatula and filtered through a double layer of sterile gauze) light and quantified in the NanoDrop 2000 spectrophotometer (Thermo Scientific).The concentration of total extracted DNA ranged from 8 to 20 ng/µL and was adjusted to 10 ng/µL for the ISSR reactions.Each 25-µL ISSR reaction was made from a mixture of 22.5 µL of PCR Mix (Invitrogen Life Technologies) (22 U/mL of recombinant Taq DNA polymerase, 22 mM of Tris-HCL, 55 mM of KCl, 1.65 mM of MgCl 2 , 220 µM of dGTP, 220 µM of dATP, 220 µM of dTTP, 220 µM of dCTP and stabilisers), 1.5 µL of each primer (10 pmol) and 1.0 µL of DNA from each isolate.The reactions were performed using the Platinum® PCR SuperMix Kit from Invitrogen TM in a Techne thermal cycler (TC-5000, Techne Inc., United Kingdom), with initial denaturation at 94˚C for 4 min, followed by 40 cycles of 94˚C for 40 seconds and an annealing temperature ranging from 45.5˚C to 46.5˚C, depending on the ISSR primer (CA1-5'ACCACACACACACACACA3', CA2-5'ATCACACACACACACACA3', CAC3 5'ATGCACCACCACCACCAC3'), with extension at 72˚C for 2 min and final extension at 72˚C for 7 min.The PCR products were stained with Gel Red®, separated by electrophoresis in agarose gel at a concentration of 1.5% and immersed in TBE buffer (0.45 M of Tris-borate and 0.01 M EDTA).The electrophoretic run was performed with 80 volts for 90 min and the PCR products were viewed in a Gel Logic 212 PRO transilluminator (Carestream Molecular Imaging Software-Version 5.0, Care- three light regimes (continuous dark, alternating light [12 h of light and 12 h of dark] and continuous light) and three temperatures (15˚C, 25˚C and 35˚C), with 12 repetitions composing an experimental area of 12.960 Petri dishes, with each Petri dish considered one repetition.Analysis of variance (F test) was performed to determine differences in mycelial growth and sporulation between isolates under the different culture conditions.Means were grouped for the analysis of the effects of the different culture media, light regimes and temperatures as well as interactions between these variables on mycelial growth and sporulation of the tangerine pathotype of A. alternata.General means were compared using the Scott-Knott test and other means were compared using Tukey's test.Statistical analysis was conducted with the aid of the SISVAR® 5.3 (Ferreira 2010) and SAS/STAT® 9.3 [25] software programs, with the level of significance set to 5% (p ≤ 0.05).Genetic diversity among the 38 isolates was determined based on the presence/absence of DNA fragments amplified by three primers and viewed in electrophoresis gels.The gel images were processed using the BioNumerics® software (version 7.1, Applied Maths, Belgium) for groups of isolates and the analysis of genetic similarity based on the Jaccard coefficient with 2% tolerance.The similarity dendogram was plotted from the arithmetic means of the groups formed in pairs of combined data (UPGMA).

Figure 1 .
Figure 1.Dendrogram with isolates of Alternaria alternata pathotype Tangerine collected in different locations, grouped according to estimates of genetic similarity obtained by the polymorphic bands generated by the ISSR (UPGMA) method.UFPB, Areia, Brazil.2016.

Table 1 .
Isolates of Alternaria alternata pathotype Tangerine used in this work.UFPB,

Table 2 .
Mean values of Mycelial Growth (MG) and Sporulation (SP) of Alternaria alternata isolates pathotype Tangerine.UFPB, Areia, Brazil, 2016.Means followed by the same letter do not differ from one another by the Scott-Knott test (p ≤ 0.05).

Table 3 .
Mean values of Mycelial Growth (MG) of Alternaria alternata isolates, pathotypeTangerine on different cultive conditions.UFPB, Areia, Brazil, 2016.The cultivation of the fungus adjusted to different light regimes (continuous dark-CD, alternating light-AL [12 hours of light and 12 hours of dark] and continuous light-CL), temperatures (15˚C, 25˚C and 35˚C) and culture media ODA (oatmeal dextrose agar-65 g of oatmeal, 20 g of dextrose, 20 g of agar and 1000 mL of SDW], PDA (potato dextrose agar-200 g of potato, 20 g of dextrose, 20 g of agar and 1000 mL SDW), LEA (leaf extract agar-400 g of citric leaves, 20 g of dextrose, 20 g of agar and 1000 mL of SDW] and V8 agar [100 mL of V8 juice, 20 g of agar, 15 g of CaCO 3 and 900 mL of SDW]).Means followed by the same letter do not differ by the Tukey test (p ≤ 0.05).Greek letter for light regimes, lowercase for culture media and upper case for temperatures.

Table 4 .
Sporulation of Alternaria alternata pathotype Tangerine in different culture conditions.UFPB, Areia, Brazil, 2016.The cultivation of the fungus adjusted to different light regimes (continuous dark-CD, alternating light-AL [12 hours of light and 12 hours of dark] and continuous light-CL), temperatures (15˚C, 25˚C and 35˚C) and culture media ODA (oatmeal dextrose agar-65 g of oatmeal, 20 g of dextrose, 20 g of agar and 1000 mL of SDW], PDA (potato dextrose agar-200 g of potato, 20 g of dextrose, 20 g of agar and 1000 mL SDW), LEA (leaf extract agar-400 g of citric leaves, 20 g of dextrose, 20 g of agar and 1000 mL of SDW] and V8 agar [100 mL of V8 juice, 20 g of agar, 15 g of CaCO 3 and 900 mL of SDW]).Means followed by the same letter do not differ by the Tukey test (p ≤ 0.05).Greek letter for light regimes, lowercase for culture media and upper case for temperatures.