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Phyllosticta citricarpa, Elsinoe fawcettii, Elsinoe australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg μL−1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg μL−1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

Poplar plantations provide important industrial feedstock in China. Red spot symptoms were observed on leaves of Populus tomentosa and P. deltoides in southeastern China. Based on morphology and molecular phylogenetic analysis, the fungus isolated from disease spots was identified as Elsinoë australis, which has been previously recognized as a pathogen of Citrus spp. and jojoba but has not been reported in China. Pathogenicity tests found that isolates from two poplar species caused red spot symptoms on leaves from different poplar species and also led to scab formation on the fruit of one hybrid citrus but not on fruit of orange, lemon, or grapefruit. The draft genome of one E. australis isolate was generated. The genetic architecture of the MAT1-1 and MAT1-2 loci of E. australis was revealed by genome sequence and long-range PCR analyses. Single isolates carried only one of two opposite mating-types was confirmed by idiomorph-specific PCR, suggesting a heterothallic mating system. Our results not only revealed a new E. australis pathotype causing poplar spot anthracnose in China but also provided its genome and mating system information.

[FRUIT QUALITIES OF CALAMANSI UNDER DIFFERENT SCAB DISEASE SEVERITIES]. Citrus scab caused by Elsinoe fawcettii dan Elsinoe australis fungi is an important disease for calamansi lime. The pathogen can result creamy-yellowish, raised, rounded, corky scabs appear on the rind surface of the fruit that reduces marketabilityof the fruit. The objective of this study was to evaluate the development and intensity of scab and their effects on the physical and chemical qualities of calamansi fruits. The observations were made on 30 fruit samples collected from farmer’s calamansi orchard for the development and intensity of disease symptoms, fruit appearances, juice content, and total soluble solids of the juice. The results showed that the pathogen began to attack on early fruit set phase. The intensity of the disease at the beginning of the observation was 11.06% and increased to 58.18% with an average rate of attack intensity of 14.54%. The disease reduced the fruit weight, fruit diameter, juice volume and total soluble solid levels.

Elsinoë fawcettii and E. australis (phylum Ascomycota) are phytopathogenic fungi causing scab diseases on citrus plants. We report here the high-quality draft genome sequences and ab initio gene predictions of two E. fawcettii strains and one E. australis strain, which differ in their host range. This genome sequence information will provide valuable resources to underpin genomic attributes for determining host range through comparative genomic analyses of citrus scab fungi.

A new disease of jojoba in Australia is described. We have demonstrated that this disease is caused by Elsinoë australis, a pathogen which is normally associated with citrus. This pathogen has not been found previously in Australia on citrus or any other crop. The fungus causes a scab on leaves and stems of jojoba and is widely distributed in eastern Australia. Although molecular analysis of the pathogen indicates that it is closely related to the natsudaidai and the sweet orange pathotypes of E. australis, glasshouse and laboratory experiments demonstrate that it is not pathogenic to a range of citrus cultivars grown in Australia. The data indicate that the isolates from jojoba represent a new pathotype of E. australis.

Sweet orange scab (SOS) is a fungal disease of citrus which is caused by Elsinoe australis. It affects the aesthetics of the fruit by forming wart-like protruded lesions on the fruit skin, and also affects the leaves which act as source of inoculum in the orchards. SOS is widespread in the different citrus species such as grapefruit (Citrus x paradisi Macfd.), sweet orange [C. sinensis (L.) Osb.], and mandarin (C. reticulata Blanco). In this study, we report the development of symptoms and changes in polyphenolic profile of ‘Rio Red’ grapefruit leaves in response to E. australis infection. Qualitative and quantitative analysis of polyphenols in ‘Rio Red’ grapefruit leaves inoculated with E. australis were conducted using high performance liquid chromatography (HPLC) at two different time points post inoculation. Development of SOS symptoms under light was recorded and categorized into four different morphological stages. Caffeic acid, luteolin-7-O glycoside, naringin, naringenin, apigenin-7-O glycoside and eriodictoyl were identified in healthy grapefruit leaves. The quantitative changes in the identified polyphenols were measured 9 days post-inoculation by comparing levels on the 9-day control versus the 9-day post-inoculated leaves. Total polyphenol levels decreased substantially in E. australis-infected ‘Rio Red’ grapefruit leaves 9 days post-inoculation. Levels of naringin, naringenin, apigenin glucoside and eriodictoyl decreased with E. australis infections indicating the pathogen’s ability to overcome some of plant’s defenses. Compared to 9-day controls, 9-day inoculated leaves had much less caffeic acid.

Molecular phylogenetic analysis, morphology and pathogenicity to citrus fruit were used to study two isolates of Elsinoe australis associated with scab-like symptoms on a fruit of Citrus australasica (finger lime) and Simmondsia chinensis (jojoba) in Australia. In addition to being associated with finger lime, the isolate from finger lime could cause scab symptoms on C. × aurantium cv. Murcott tangor in pathogenicity tests, but could not cause scab symptoms on the other orange, mandarin, lemon or grapefruit tested. Pathogenicity tests also support previous studies showing the isolate from jojoba could not produce symptoms on fruit of C. natsudaidai. Based on the findings of this study, two novel pathotypes of E. australis are designated from Australia; namely the Finger Lime (FL) pathotype associated with finger lime, and the Jojoba Black Scab (JBS) pathotype associated with black scab of jojoba. The significance of these novel E. australis pathotypes on market access and biosecurity issues for citrus are briefly discussed.

Elsinoe fawcettii and E. australis are important pathogens of citrus. Both species are known to produce red or orange pigments, called elsinochrome. Elsinochrome is a nonhost-selective phytotoxin and is required for full fungal virulence and lesion formation. This article discusses the taxonomy, epidemiology, genetics and pathology of the pathogens. It also provides a perspective on the cellular toxicity, biosynthetic regulation and pathological role of elsinochrome phytotoxin. Taxonomy: Elsinoe fawcettii (anamorph: Sphaceloma fawcettii) and E. australis (anamorph: S. australis) are classified in the Phylum Ascomycota, Class Dothideomycetes, Order Myriangiales and Family Elsinoaceae. Host range: Elsinoe fawcettii causes citrus scab (formerly sour orange scab and common scab) on various species and hybrids in the Rutaceae family worldwide, whereas E. australis causes sweet orange scab, primarily on sweet orange and some mandarins, and has a limited geographical distribution. Disease symptoms: Citrus tissues infested with Elsinoe often display erumpent scab pustules with a warty appearance. Toxin production: Elsinochrome and many perylenequinone-containing phytotoxins of fungal origin are grouped as photosensitizing compounds that are able to absorb light energy, react with oxygen molecules and produce reactive oxygen species, such as superoxide and singlet oxygen. Elsinochrome has been documented to cause peroxidation of cell membranes and to induce rapid electrolyte leakage from citrus tissues. Elsinochrome biosynthesis and conidiation are coordinately regulated in E. fawcettii, and the environmental and physiological inducers commonly involved in both processes have begun to be elucidated.

Elsinoe fawcettii causes citrus scab and E. australis causes sweet orange scab and there are different pathotypes of each species. Citrus scab is widely distributed, whereas sweet orange scab is limited mostly to southern South America. In 2010, E. australis was detected by PCR in Texas for the first time in the USA and subsequently found in commercial areas of Texas. In this study, cultures were obtained in Texas mostly from diseased fruit with symptoms similar to "late-season windscar" and identified as the Natsudaidai pathotype of E. australis by sequencing of the PCR amplification products and sequencing of the translation elongation factor and ITS regions. Inoculations of detached leaves and fruit of grapefruit, sweet orange, and tangerine produced scab-like symptoms on all organs and species. The fungus was re-isolated from the inoculated organs and PCR tests on re-isolated fungal colonies and lesions confirmed the presence of E. australis. The disease represents sweet orange scab of citrus with a new scab disease symptomatology, but the host range, ecology and epidemiology of this pathotype are still not completely understood. Thus far, Natsudaidai pathotype has been reported only from South Korea.

Genetic differences among three citrus scab pathogens were investigated. The three pathogens were (i) the cosmopolitan Elsinoe fawcettii causing citrus scab, (ii) E. australis from South America causing sweet orange scab, and (iii) Sphaceloma fawcettii var. scabiosa from Australia causing Tryon's scab. In a companion study, we were unable to differentiate these taxa morphologically, but were able to differentiate E. fawcettii, E. australis, and S. fawcettii var. scabiosa pathologically. Two pathotypes of E. fawcettii from Florida and two pathotypes from Australia were distinguished on the basis of host range. Restriction analysis of the amplified internal transcribed spacer (ITS) of ribosomal DNA with several endonucleases and sequence analysis of the ITS readily differentiated E. australis from E. fawcettii and S. fawcettii var. scabiosa. Random amplified polymorphic DNA (RAPD) analysis indicated that E. fawcettii isolates from Australia and from Florida were more closely related to each other than to E. australis isolates from Argentina. However, all Australian isolates were separable from all Florida isolates by their RAPD profiles. There was a good correspondence between the RAPD profiles and the two pathotypes identified in Australia. Molecular analysis will be a rapid, useful tool in identifying exotic types of citrus scab on shipments of fruit and help reduce introductions of these types into new areas.

Three scab diseases have been described on citrus: (i) citrus scab caused by Elsinoe fawcettii, which is cosmopolitan in humid citrusgrowing areas; (ii) Tryon's scab caused by Sphaceloma fawcettii var. scabiosa, which occurs in Australia and elsewhere; and (iii) sweet orange scab caused by E. australis, which occurs primarily in southern South America. In this study, we compared isolates from Australia, Argentina, and Florida (United States). Neither colony color nor conidial size or shape could be used to distinguish the three scab fungi on citrus. A detached-leaf assay was developed to compare pathogenicity of isolates on rough lemon, sour orange, and grapefruit. All isolates from Australia produced scab only on rough lemon, whereas all isolates from Florida produced scab on rough lemon and grapefruit and a portion of these also produced scab on sour orange. E. australis isolates from Argentina did not affect leaves of any species. One E. fawcettii isolate from Argentina produced the same reaction as Australian isolates, and two produced the same reaction as the Florida group that affected rough lemon, grapefruit, and sour orange. In greenhouse inoculations, all Australian isolates infected rough lemon, lemon, and Rangpur lime; all but a few also infected Cleopatra mandarin. In greenhouse inoculations in Florida, all isolates infected rough lemon and Cleopatra mandarin and some isolates infected sour orange. Although E. australis is not readily differentiated from E. fawcettii morphologically, we concluded that they are separate species on the basis of pathogenicity and molecular analysis. S. fawcettii var. scabiosa could be differentiated from E. fawcettii only by host range. We recognized four pathotypes of E. fawcettii: the Broad Host Range and Narrow Host Range pathotypes originally described in Florida, the Tryon's pathotype (formerly S. fawcettii var. scabiosa), and a new Lemon pathotype from northern Australia.

A description is provided for Elsinoe australis . Information is included on the disease caused by the organism, its transmission, geographical distribution, and hosts. HOSTS: Sweet orange, lemon, mandarin, tangerine, satsuma, orange, kumquat ( Fortunella margarita ), lime, grapefruit and pointed leaf papeda ( Citrus hystrix ) (19, 366; Brun, 1971). Sour oranges generally resistant to attack but symptoms have been occasionally noted (20, 110; 35, 544). DISEASE: Sweet orange scab. Symptoms mainly on fruits, less frequent on or absent from leaves and twigs. Only young tissues are attacked (22, 354). Young fruits may become distorted. GEOGRAPHICAL DISTRIBUTION: Largely confined to South America (Argentina, Bolivia, Brazil, Paraguay), although a single report occurs from Sicily (37, 234). Symptoms of scab on citrus in the Dominican Republic (CMI Map 55, ed. 2, 1965) and New Caledonia (36, 711) initially attributed to E. australis are now thought to have been due respectively to E. fawcettii (CMI Descript. 438) and E. fawcettii var. scabiosa (CMI Descript. 437) (41, 85; 51, 3250; CMI Map 55, ed. 2, 1965). TRANSMISSION: Presumably by wind and rain splash. Old lesions on fruits, leaves and twigs are the probable source of infection at the start of each season (Bitancourt & Jenkins, 1937a).