Diaporthe species are important pathogens of a wide range of hosts including many economically important crops. Diaporthe citri is generally accepted to cause melanose of Citrus fruits, leaves and shoots; stem-end rot of fruits and gummosis of perennial branches. In this study, 147 strains of Diaporthe were isolated from diseased leaves, twigs, fruits and branches of Citrus from Shaanxi, Zhejiang, Guangxi, Fujian, Guangdong, Jiangxi, Hunan, Chongqing and Shanghai and identified by morphology and molecular analysis of nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (EF 1-α), tubulin (TUB) and calmodulin (CAL) genes. All strains from Citrus clustered in three clades. Diaporthe citri was the predominant species and is compared with strains from Florida, USA, where this species was first collected and described. Two new taxa differing from D. citri are described as D. citriasiana and D. citrichinensis in this paper. Inoculation tests indicate that all three species are able to cause stem-end rot of fruits, while only D. citri could cause melanose on leaves.

The microbiome associated with crop plants has a strong impact on their health and productivity. Candidatus Liberibacter asiaticus (Las), the bacterial pathogen responsible for Huanglongbing (HLB) disease, lives inside the phloem of citrus plants including the root system. It has been suggested that Las negatively affects citrus microbiome. On the other hand, members of citrus microbiome also influence the interaction between Las and citrus. Here, we report the isolation and characterization of multiple putative beneficial bacteria from healthy citrus rhizosphere. Firstly, six bacterial strains showing antibacterial activity against two bacteria closely related to Las: Agrobacterium tumefaciens and Sinorhizobium meliloti were selected. Among them, Burkholderia metallica strain A53 and Burkholderia territorii strain A63 are within the β-proteobacteria class, whereas Pseudomonas granadensis strain 100 and Pseudomonas geniculata strain 95 are within the γ-proteobacteria class. Additionally, two gram-positive bacteria Rhodococcus jialingiae strain 108 and Bacillus pumilus strain 104 were also identified. Secondly, antimicrobial activity against three fungal pathogens: Alternaria alternata, Colletotrichum acutatum, Phyllosticta citricarpa, and two oomycetes: Phytophthora nicotianae and Phytophthora palmivora. Four bacterial strains Burkholderia territorii A63, Burkholderia metallica A53, Pseudomonas geniculata 95, and Bacillus pumilus 104 were shown to have antagonistic activity against the citrus root pathogen Phytophthora nicotianae based on dual culture antagonist assays and compartmentalized petri dish assays. The four selected bacteria were sequenced. Genes involved in phosphate solubilization, siderophore production and iron acquisition, volatile organic compound production, osmoprotection and osmotic tolerance, phytohormone production, antagonism, and nutrient competition were predicted and discussed related to the beneficial traits.

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 10¹ to 10⁵ conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 10⁵ conidia/ml and 40 μM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC₅₀) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC₅₀ values were observed (R² = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.

Phyllosticta citricarpa, the citrus black spot pathogen, was first identified in Florida in March 2010. Subsequently, this pathogen has become established in Florida but can be easily confused with the endemic nonpathogenic citrus endophyte P. capitalensis. In this study, the mating-type (MAT) loci of P. citricarpa and P. capitalensis were identified via draft genome sequencing and were characterized at the structural and sequence levels. P. citricarpa was determined to have an idiomorphic, heterothallic MAT locus structure, whereas P. capitalensis was found to have a single MAT locus consistent with a homothallic mating system. A survey of P. citricarpa isolates from Florida revealed that only the MAT1-2 idiomorph existed in the Floridian population. In contrast, isolates collected from Australia exhibited a 1:1 ratio of MAT1-1 and MAT1-2 isolates. Development and analysis of simple sequence repeat markers revealed a single multilocus genotype (MLG) in the Floridian population (n = 70) and 11 MLG within the Australian population (n = 24). These results indicate that isolates of P. citricarpa from Florida are likely descendent from a single clonal lineage and are reproducing asexually. The disease management focus in Florida will need to be concentrated on the production and dispersal of pycnidiospores.

Chemical control, based on copper and quinone outside inhibitor (QoI) fungicides, has been essential for the management of brown spot of citrus, caused by Alternaria alternata. However, QoI control failures were detected recently in Florida. From 2008 to 2012, 817 monoconidial isolates of A. alternata from 46 citrus orchards were examined for sensitivity to azoxystrobin (AZ) and pyraclostrobin (PYR). Of the isolates, 57.6% were resistant to both fungicides, with effective concentration to inhibit 50% growth (EC₅₀) values greater than 5 μg/ml for AZ and 1 μg/ml for PYR. The mean EC₅₀ values for sensitive isolates were 0.139 and 0.020 μg/ml for AZ and PYR, respectively. The EC₅₀ values of both fungicides were highly correlated (P < 0.0001), indicating cross resistance. The proportion of resistant isolates differed significantly (P < 0.0001) among cultivars and with QoI application frequency (P < 0.0001). However, resistance was not significantly related (P = 0.364) to disease severity in the field (low, moderate, and high) or isolate virulence (P = 0.397). The molecular basis for QoI resistance was determined for a subset of 235 isolates using polymerase chain reaction restriction fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutation G143A. Based on the presence of one or two introns, isolates were classified as profile I and profile II, respectively. The resistance frequency was significantly higher (P < 0.0001) in isolate profile II, suggesting a higher selection pressure for resistant population profile II.

In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 μm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or carrot agar and incubated with 12 h of light and dark at 24°C. Cultures that grew colonies within a week were discarded. Fourteen single-spore cultures were obtained from the isolates that grew slower than the Guignardia mangiferae reference cultures, although pycnidia formed more rapidly in the G. mangiferae cultures (1). No sexual structures were observed. Cultures on half-PDA were black and cordlike with irregular margins with numerous pycnidia, often bearing white cirrhi after 14 days. Conidia (7.1 to 7.8 × 10.3 to 11.8 μm) were hyaline, aseptate, multiguttulate, ovoid with a flattened base surrounded by a hyaline matrix (0.4 to 0.6 μm) and a hyaline appendage on the rounded apex, corresponding to published descriptions of G. citricarpa (anomorph Phyllosticta citricarpa) (1). A yellow pigment was seen in oatmeal agar surrounding G. citricarpa, but not G. mangiferae colonies as previously reported (1,2). DNA was extracted from lesions and cultures and amplified with species-specific primers (2). DNA was also extracted from G. mangiferae and healthy citrus fruit. The G. citricarpa-specific primers produced a 300-bp band from fruit lesions and pure cultures. G. mangiferae-specific primers produced 290-bp bands with DNA from G. mangiferae cultures. The internally transcribed spacer (ITS) of the rRNA gene, translation-elongation factor (TEF), and actin gene regions were sequenced from G. citricarpa isolates and deposited in GenBank. These sequences had 100% homology with G. citricarpa ITS sequences from South Africa and Brazil, 100% homology with TEF, and 99% homology with actin of a Brazilian isolate. Pathogenicity tests with G. citricarpa were not done because the organism infects immature fruit and has an incubation period of at least 6 months (3). In addition, quarantine restrictions limit work with the organism outside a contained facility. To our knowledge, this is the first report of black spot in North America. The initial infested area was ~57 km². The disease is of great importance to the Florida citrus industry because it causes serious blemishes and significant yield reduction, especially on the most commonly grown ‘Valencia’ sweet orange. Also, the presence of the disease in Florida may affect market access because G. citricarpa is considered a quarantine pathogen by the United States and internationally. References: (1) R. P. Baayen et al. Phytopathology 92:464, 2002. (2) N. A. Peres et al. Plant Dis. 91:525, 2007 (3) R. F. Reis et al. Fitopath Bras. 31:29, 2006.