RESISTANCE TO ALTERNARIA BROWN SPOT OF NEW CITRUS HYBRIDS

Alternaria brown spot (ABS) disease is caused by the fungus of Alternaria alternata f. sp. citri, which causes injury in leaves, branches and fruits of citrus. The action of the pathogen is directly related to the presence of toxin receptors in susceptible genotypes. The objective of this study was to characterize a population of citrus hybrids obtained from controlled crosses between Pêra de Abril sweet orange and the hybrid of Murcott tangor x Pêra sweet orange (TM x LP 163) for response to ABS through the in vitro inoculation of fungal spores in young detached leaves. The fungus was isolated from the lesions of Murcott tangor fruits that exhibited ABS symptoms. Two hundred thirty-five hybrids were evaluated, and 70 (30%) showed different levels of disease symptoms on detached leaves after 72 hours of inoculation with the fungus, and 165 (70%) were asymptomatic. The frequency of segregation observed (165R:70S) and high level of heritability (hg = 0.91) suggest that few genes may be involved in controlling the inheritance of ABS resistance in citrus.

ABSTRACT-Alternaria brown spot (ABS) disease is caused by the fungus of Alternaria alternata f. sp.citri, which causes injury in leaves, branches and fruits of citrus.The action of the pathogen is directly related to the presence of toxin receptors in susceptible genotypes.The objective of this study was to characterize a population of citrus hybrids obtained from controlled crosses between Pêra de Abril sweet orange and the hybrid of Murcott tangor x Pêra sweet orange (TM x LP 163) for response to ABS through the in vitro inoculation of fungal spores in young detached leaves.The fungus was isolated from the lesions of Murcott tangor fruits that exhibited ABS symptoms.Two hundred thirty-five hybrids were evaluated, and 70 (30%) showed different levels of disease symptoms on detached leaves after 72 hours of inoculation with the fungus, and 165 (70%) were asymptomatic.The frequency of segregation observed (165R:70S) and high level of heritability (h 2 g = 0.91) suggest that few genes may be involved in controlling the inheritance of ABS resistance in citrus.Index terms: Alternaria alternata, genetic breeding, Murcott tangor, Pêra de Abril, sweet orange.
ABS was first identified in Australia in Emperor mandarin (C.reticulata Blanco) around 1903, and the disease soon spread to other places in the world; it is now present in several countries, such as Turkey, Spain, Italy, South Africa, the USA, China, Brazil and Argentina (STUART et al., 2009;CUENCA et al., 2013CUENCA et al., , 2016;;HUANG et al., 2015).In Brazil, the disease was recorded in Rio de Janeiro in 2001 (GOES et al., 2001) in Dancy mandarin, and it later spread to other states, such as Minas Gerais, Rio Grande do Sul and São Paulo (SPOSITO et al., 2003;STUART et al., 2009).
ABS is mainly disseminated through conidia (asexual spores) of the fungus, and the process of colonization involves the production of host-specific toxin (HST) (TSUGE et al., 2013).ABS symptoms occur in leaves, branches and fruits.The symptoms initially appear as small lesions that expand by the action of the toxin and cause intense defoliation during periods of increased infection (TIMMER et al., 2003).The toxin type ACT produced by A. alternata f. sp.citri is widely described in the literature and is specific to a particular host range (KOHMOTO et al., 1991;PEEVER et al., 1999), including mandarins and some hybrids.
In fine branches, small cortical lesions occur, with or without a chlorotic halo.In mature fruits, necrotic spots 1 to 10 mm in diameter occur, and the external quality of these fruits is severely reduced, depreciating them for consumption (TIMMER et al., 2003;TSUGE et al., 2013;BASSIMBA et al., 2014).
The resistance of citrus plants to ABS has been observed in species of citrus such as sweet orange (STUART et al., 2009), willow leaf mandarins and mandarins like Fremont, Thomas, Clementina and Cravo (REIS et al., 2007;SOUZA et al., 2009;PACHECO et al., 2012).
Few studies have investigated the origin of resistance and susceptibility of citrus genotypes to ABS.Susceptibility is likely simple and controlled by dominant inheritance (DALKILIC et al., 2005;GULSEN et al., 2010;CUENCA et al. 2013CUENCA et al. , 2016)).Thus, the "A" allele would be dominant for susceptibility, and "a" would be recessive for disease resistance.
Studies conducted using populations of triploid hybrids (CUENCA et al., 2013)

MATERIAL AND METHODS
A population of 235 hybrids was obtained in 2010 from controlled crossings between Pêra de Abril sweet orange (C.sinensis) and TM x LP 163.The latter is a hybrid between Murcott tangor (C.reticulata x C. sinensis) x Pêra sweet orange (C.sinensis), produced in 2010.The population was available for evaluation in the greenhouse at the Sylvio Moreira Citrus APTA Center of the Agronomic Institute (IAC).
Isolates of A. alternata f. sp.citri were obtained from injured tissues of Murcott tangor fruits collected from plants grown in the field at the Sylvio Moreira APTA Citrus Center of the Agronomic Institute (IAC).Leaf tissue with characteristic lesions was cut into small pieces with a scalpel.Surface disinfection was then performed by immersion in commercial ethyl alcohol (70%) for 1 minute followed by immersion in 2% sodium hypochlorite for 2 minutes, after which the samples were transferred to Petri dishes containing potato dextrose agar medium (200 g potato, 20 g dextrose, 15 g L -1 agar).To prevent the growth of other fungal contaminants, fungicide with carbendazim (640 mg L -1 ) as the active ingredient was added.To avoid the growth of bacteria, 50 µg mL -1 tetracycline was added to the culture medium.The plates were maintained under a photoperiod of 12 hours and a temperature of 25 °C (SASSERON, 2008).
After seven days, media composed of 30 g of calcium carbonate, 20 g of sucrose and 20 g of agar was prepared to a volume of 1 L and poured into Petri plates.Fifty 5-mm-wide discs with mycelial tissue growth were then transferred to this medium to induce sporulation.This material was maintained at 27 °C for 5 days under a 12-hour photoperiod.The identity of the isolates was confirmed via the optical microscopy of asexual structures.
For in vitro inoculation, young leaves were collected from all hybrids and their parents.The ABS-susceptible Dancy mandarin and Murcott tangor varieties and the ABS-resistant Fremont mandarin variety (AZEVEDO et al., 2010) served as control genotypes.Three leaves per inoculated plant were placed in a Petri dish with moistened filter paper and cotton, which were previously sterilized.The leaves were sprayed with 2 mL of a suspension containing the spores at a concentration of 10 5 conidia mL -1 according to the methodology described by Peever et al. (1999) and Canihos et al. (1999).The experiment was conducted in a growth chamber at 27 °C under a 12-hour photoperiod.The experimental design was completely randomized with three replicates per genotype, and each replicate was represented by one Petri dish.
The evaluations of symptoms caused by the fungus were performed at 24, 48 and 72 hours after inoculation by observing the presence of typical symptoms of the disease.The determination of severity was assessed for a subset of three leaves per treatment using a diagrammatic scale including nine levels of severity in leaves expressed as the percentage of infected area (0, 0.3, 3.5, 8.0, 15, 34, 61, 80, 90 and 97%) (MARTELLI et al., 2016).
To better understand the disease progress over time, it was calculated the area under the disease progress curve (AUDPC).Based on the results, a representative histogram of the population in relation to ABS severity was constructed using the software R.
The severity of the disease data at the three times of evaluation was used to calculate the AUDPC as follows: where y1 and y2 are two consecutive assessments carried out at times t1 and t2, respectively.This calculation is a statistical analysis that allows the verification of the behavior of the disease by the progress curve (BERGAMIN, 1995) from mathematical models used previously.
The AUDPC values were used to calculate genetic parameters such as variation (genetic and environmental) and heritability.For this, the program SELEGEN -REML/BLUP was used (RESENDE and SILVA, 2014).

RESULTS AND DISCUTION
Table 1 shows the severity values assessed using the diagrammatic scale and the AUDPC.In the first evaluation, 24 hours after the inoculation of detached leaves in vitro, leaf lesions were observed in 54 hybrids (22.9%) and in varieties used as positive controls (Murcott tangor and Dancy mandarin), as expected (Figure 1).After 48 hours of inoculation, 54 hybrids were considered symptomatic, and 14 new hybrids showed symptoms typical of the disease.At this evaluation stage, a higher percentage of affected leaf area in the leaves where symptoms started was observed at 24 hours.Values ranging from 0.3% (24 hours) to 61% (48 hours) of the leaf area with symptoms were also observed.Forty-eight hours after inoculation, a 22.98% (24 hours) to 28.94% increase in the percentage of individuals showing symptoms was observed.
After 72 hours, an increase in the area of necrosis on the leaves in which symptoms began within 24 hours was observed, and the hybrid PAx163-3 showed maximum severity, as assessed by the diagrammatic scale (97%).Throughout the population, 165 (70%) asymptomatic individuals were observed, and 70 (30%) other individuals showed different levels of symptoms (Figure 2).Studies on inheritance of ABS resistance in citrus were based on crosses between mandarins varieties (CUENCA et al., 2013) and between mandarin and sweet orange (CUENCA et al., 2016).These studies stated that the inheritance of ABS resistance in citrus is controlled by a single recessive allele (DALKILIC et al., 2005;GULSEN et al., 2010;CUENCA et al., 2013).Thus, the "A" allele would be dominant for susceptibility, and "a" would be recessive for disease resistance (CUENCA et al., 2013(CUENCA et al., , 2016)).Therefore, segregation is expected in progeny arising from crosses between resistant and heterozygous ABS-susceptible parents or even between two heterozygous ABS susceptible ones.In the present work, the parents Pêra de Abril sweet orange and DOI 10.1590/0100-29452017 613 Jaboticabal -SP K. AP.F. de CAMPOS et al.
the hybrid TM x LP 163, were asymptomatic, 165 (70%) asymptomatic individuals of the progeny were observed and 70 (30%) individuals showed different levels of symptoms (Figure 2).Thus, the segregation observed in F 1 plants contrasts with that reported before (DALKILIC et al., 2005;GULSEN et al., 2010;CUENCA et al., 2013CUENCA et al., , 2016) ) and suggested that two genes were involved in genetic control of this trait, rather than a single gene.Then, we suggested that due to a complete dominance for both gene pairs, only if both kinds of dominant alleles were present the susceptible phenotype appears.When one gene is homozygous recessive or both genes are homozygous recessive, the susceptible phenotype is hidden.Further studies should be conducted to elucidate the genetic mechanisms involved in sweet orange or in other varieties that do not respond to a HST produced by the pathogen.
High heritability (h 2 g = 0.91) and genotypic variability expressed by the genotypic variation coefficient (Cvgi = 228.25)(Table 2) were observed in the present work, indicating that the selection of resistant plants in the progeny can be successful.Namely, the hybrids have adequate genetic variability for selection.
TABLE 1-Severity (%) and the area under the disease progress curve (AUDPC) of alternaria brown spot evaluated after inoculation with A. alternata f. sp.citri in hybrids, parents and citrus varieties.Legend: h 2 g heritability of individual parcels in the broad sense, ie, the genotypic effects; CVgi (%): genotypic coefficient of variation in percent; CVe (%) environmental variation coefficient as a percentage; Mean: overall mean of the experiment.
BROWN SPOT OF NEW CITRUS HYBRIDS
Means followed by the same letters in the column (lower) and in the line (capital) do not differ by the Scott-Knott test (p> 0.05).

TABLE 2 -
Estimated genetic parameters based on AUDPC values of 235 F 1 hybrid progeny crossing between Pêra de Abril sweet orange vs TM x LP 163.