Genetic Diversity and Population Structure of Moroccan Isolates Belong to Alternaria spp. Causing Black Rot and Brown Spot in Citrus

Alternaria alternata is one of the most important fungi causing various diseases on citrus worldwide. In Morocco, Alternaria black rot (ABR) and Alternaria brown spot (ABS) are two major diseases causing serious losses in commercial cultivars of citrus. The aim of the present work was to study the genetic diversity and the population structure of isolates belonging to sect. Alternaria obtained from infected citrus fruits, collected from seven provinces at different locations in Morocco (markets, packinghouses, and orchards). Forty-five isolates were analyzed by sequence-related amplified polymorphism (SRAP) markers, and cluster analysis of DNA fragments was performed using UPGMA method and Jaccard coefficient. Cluster analysis revealed that isolates were classified in four distinct groups. AMOVA revealed also a large extent of variation within sect. Alternaria isolates (99%). The results demonstrate that no correlation was found among SRAP pattern, host, and geographical origin of these isolates. Population structure analyses showed that the Alternaria isolates from the same collection origin had almost a similar level of admixture.

Comparison of Four Systems to Test the Tolerance of ‘Fortune’ Mandarin Tissue Cultured Plants to Alternaria alternata

Alternaria brown spot is a severe disease that affects leaves and fruits on susceptible mandarin and mandarin-like cultivars, and is produced by Alternaria alternata. Consequently, there is an urge to obtain new cultivars resistant to A. alternata, and mutation breeding together with tissue culture can help shorten the process. However, a protocol for the in vitro selection of resistant citrus genotypes is lacking. In this study, four methods to evaluate the sensitivity to Alternaria of mandarin ‘Fortune’ explants in in vitro culture were tested. The four tested systems consisted of: (1) the addition of the mycotoxin, produced by A. alternata in ‘Fortune’, to the propagation culture media, (2) the addition of the A. alternata culture filtrate to the propagation culture media, (3) the application of the mycotoxin to the intact shoot leaves, and (4) the application of the mycotoxin to the previously excised and wounded leaves. After analyzing the results, only the addition of the A. alternata culture filtrate to the culture media and the application of the mycotoxin to the wounded leaves produced symptoms of infection. However, the addition of the fungus culture filtrate to the culture media produced results, which might indicate that, in addition to the mycotoxin, many other unknown elements that can affect the plant growth and behavior could be found in the fungus culture filtrate. Therefore, the application of the toxin to the excised and wounded leaves seems to be the most reliable method to analyze sensitivity to Alternaria of ‘Fortune’ explants cultured in vitro.

Chromosome-scale genome sequence of Alternaria alternata causing Alternaria brown spot of citrus

Alternaria brown spot (ABS) caused by Alternaria alternata is an economically important fungal disease of citrus worldwide. The ABS pathogen A. alternata tangerine pathotype can produce a host-specific ACT toxin, which is regulated by ACT toxin gene cluster located in the conditionally dispensable chromosome (CDC). Previously, we have assembled a draft genome of A. alternata tangerine pathotype strain Z7, which comprises 165 contigs. In this study, we report a chromosome-level genome assembly of A. alternata Z7 through the combination of Oxford nanopore sequencing and Illumina sequencing technologies. The assembly of A. alternata Z7 had a total size of 34.28 Mb, with a GC content of 51.01% and contig N50 of Mb. The genome is encompassed 12067 protein-coding genes, 34 rRNAs, and 107 tRNAs. Interestingly, A. alternata Z7 is composed of 10 essential chromosomes (ECs) and 2 conditionally dispensable chromosomes (CDCs), which is consistent with the experimental evidences of pulsed-field gel electrophoresis (PFGE). To our best knowledge, this is the first chromosome-level genome assembly of A. alternata. In addition, a database for citrus-related Alternaria genomes has been established to provide public resources for the sequences, annotation and comparative genomics data of Alternaria species. The improved genome sequence and annotation at the chromosome level is a significant step toward a better understanding of the pathogenicity of A. alternata. The database will be updated regularly whenever the genomes of newly isolated Alternaria species are available. The citrus-related Alternaria genomes database is open accessible through http://www.zjudata.com/alternaria/blast.php.

Induction and selection of mandarin mutants with fruits containing low number of seeds.

The Brazilian citrus industry has a worldwide presence for production and export of sweet orange juice, but it has little contribution to the production of fruits for the fresh fruit market. One requirement of this market is the production of seedless fruits. The Fremont IAC 543 mandarin produces fruits with good commercial qualities, large numbers of seeds (10-12), and plants with resistance to Alternaria brown spot (ABS), an important disease present in several countries. The objective of this work was to induce and select mutants of Fremont IAC 543 mandarin with seedless fruits or fruits with a low number of seeds, using gamma-ray induced mutagenesis. In vivo buds were irradiated with doses of 20 and 30 Gy of gamma-rays. After irradiation and grafting of 2000 in vivo buds with each mutagenic dose, 4000 plants were produced and planted in an experimental field. During development of these plants, they were pruned several times allowing only the development of M1V4 branches or more advanced ones (without new grafting). A total of 32 branches were selected during the harvesting period because they produced seedless fruits and nine mutant clones were selected after another vegetative multiplication. Fruit and juice qualities, including seed number of the fruits, were evaluated in a further experiment including six mutants and a control. The results obtained showed that all mutants produced fruits with a lower number of seeds (between 3.7 and 9.1 seeds per fruit) in relation to the control (22.0 seeds per fruit), but without the existence of other alterations (fruit metric and chemical characteristics of the juice). All selected mutants (nine) are participating in advanced agronomic evaluation experiments, with a greater number of replicates and several local checks, in order to evaluate commercial yield, presence of chimeras, disease resistance and organoleptic quality of the fruits.

Disease Resistant Citrus Breeding Using Newly Developed High Resolution Melting and CAPS Protocols for Alternaria Brown Spot Marker Assisted Selection

Alternaria alternata is a fungus that causes a serious disease in susceptible genotypes of citrus, particularly in mandarins. The Alternaria citri toxin (ACT) produced by the pathogen induces necrotic lesions on young leaves and fruits, defoliation and fruit drop. Here, we describe two methods of marker-assisted selection (MAS) that could be used for the early identification of Alternaria brown spot (ABS)-resistant mandarin hybrids. The first method is based on a nested PCR coupled to high resolution melting (HRM) analysis at the SNP08 locus, which is located at 0.4 cM from the ABS resistance locus, and was previously indicated as the most suitable for the selection of ABS-resistant hybrids. The method was validated on 41 mandarin hybrids of the CREA germplasm collection, and on 862 progenies generated from five crosses involving different susceptible parents. Four out of five populations showed Mendelian segregation at the analyzed locus, while a population involving Murcott tangor as male parent showed distorted segregation toward the susceptible hybrids. The second method is based on a cleaved amplified polymorphic sequences (CAPS) marker that was developed using the same primers as the nested PCR at the SNP08 locus, coupled with BccI restriction enzyme digestion. To verify the reliability of the two genotyping methods, in vitro leaf phenotyping was carried out by inoculating A. alternata spores onto young leaves of 101 hybrids, randomly chosen among the susceptible and resistant progenies. The phenotyping confirmed the SNP08 genotyping results, so the proposed method of selection based on HRM or CAPS genotyping could be routinely used as an alternative to KBioscience competitive allele specific polymerase chain reaction (KASPar) single nucleotide polymorphism (SNP) genotyping system to improve citrus breeding programs. While the study confirmed that the SNP08 marker is a reliable tool for MAS of new citrus hybrids with different genetic backgrounds, it also identified a small group of genotypes where the resistance mechanism requires further investigation.

Response of citrus hybrids to Alternaria alternata inoculation

Citrus orchards have some limitations, such as the occurrence of phytosanitary problems. Alternaria brown spot (ABS) is caused by fungus Alternaria alternata, which affects several parts of the plant by producing a host-specific toxin, known as ACT. ABS is a limiting factor in orchards due to the susceptibility of most planted cultivars: ‘Murcott’ tangor and ‘Ponkan’ tangerine. The selection of varieties resistant/tolerant to the disease has economic importance. Therefore, the aim of this experiment was to evaluate the response to A. alternata inoculation in a population of ‘Murcott’ tangor vs ‘Pera’ sweet orange hybrids. Leaves of 2-3 centimeters in length of ‘Murcott’ tangor, ‘Pera’ sweet orange, ‘Ponkan’, ‘Dancy’, ‘Fremont’ tangerine and 198 hybrids were collected. For in vitro inoculation, monosporic A. alternata culture at concentration of 105 conidia mL-1 was used. Inoculated leaves were stored in humid chamber. After 24, 48 and 72 hours of inoculation, leaf lesions were evaluated following a diagrammatic scale. The results obtained showed that most hybrids from the crossing of ‘Murcott’ tangor vs ‘Pera’ sweet orange are susceptible to ABS. However, 44 are resistant and ten are tolerant. Among ABS-tolerant hybrids, some have phenotype similar to that of cultivated and commercialized hybrids.

2020–2021 Florida Citrus Production Guide: Alternaria Brown Spot

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