Crop loss, aetiology, and epidemiology of citrus black spot in Ghana

Citrus Black Spot (CBS), caused by Guignardia citricarpa, was detected for the first time in Ghana and in West Africa. The disease was first observed in the Eastern Region in 1999 with typical disease symptoms including hard spot, virulent spot and false melanose were observed on several citrus species. A survey revealed that the disease has reached epidemic levels in the citrus-producing areas of the Eastern and Ashanti regions and is spreading rapidly within these areas and to other regions of the country. Currently, CBS is the most important fruit disease of citrus in Ghana, causing about 22% crop loss. Although the disease does not cause postharvest decay and the internal quality of the fruit is not affected, significant amounts of blemished fruit are discarded at the markets. Disease incidence and severity was found to be higher on mature than on young citrus trees. Pycnidia were found on fruit with hard spot symptoms, and pycnidia and pseudothecia typical of Guignardia spp. were found on decomposing leaves. Two species, G. citricarpa and G. mangiferae, were isolated from 15% of the samples collected and identified using the Oatmeal Agar test and by PCR with species-specific DNA primers. Isolates of G. citricarpa produced CBS symptoms after 80 to 233?days on 75% of the artificially inoculated young fruit of Valencia Late sweet orange. The fungus was re-isolated from symptomatic, inoculated fruit completing Koch’s postulates. Isolates of the endophyte G. mangiferae did not induce symptoms in the pathogenicity tests. In epidemiological studies, infections were detected from November to February for the minor cropping season and from May to November for the major season. Fruit of Valencia Late sweet orange were susceptible to G. citricarpa infection for up to 7?months after petal fall. Knowledge of the disease cycle in Ghana will improve methods for disease control.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

Citrus black spot intensity and yield losses on sweet orange are affected by Phyllosticta citricarpa inoculum concentration and fruit developmental stage

Citrus black spot (CBS) symptom expression on sweet oranges has been reported to be affected by the fruit developmental stage and inoculum concentration in greenhouse conditions. However, there is little information concerning the period in which the fruit is susceptible in commercial orchards. This study assessed the influence of fruit age and inoculum concentration of Phyllosticta citricarpa on CBS intensity and fruit drop in the field. Two field trials were conducted in commercial ‘Valencia’ orchards in São Paulo state, Brazil. Fruit were inoculated from October (petal fall stage) to July (c.6.5 cm diameter), with 10¹, 10³ and 10⁵ pycnidiospores/ml. CBS symptoms and fruit drop were observed in higher levels for fruit inoculated from October to February than from March to July. In both trials, when fruit were inoculated 10 times from October to July or single-inoculated in November or December, high CBS severities of 11%–22% were observed and the proportion of fruit remaining on the tree was 48%–77%; in contrast, noninoculated fruit had severity below 1.1% and 90% remained on the tree. Inoculations in November or December of green fruit with 1.5–3.0 cm diameter using 10⁵ pycnidiospores/ml were associated with higher CBS intensity and fruit drop and shorter incubation period than inoculations with lower concentrations. This work provides a better understanding of fruit stages and P. citricarpa inoculum concentrations most related to symptom expression and losses under natural conditions and may be helpful for improvement of CBS management in the São Paulo citrus belt.     

Enhanced resistance to citrus canker in transgenic sweet orange expressing the sarcotoxin IA gene

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subp. Citri (Xcc), is a serious disease reported in most citrus-producing areas around the world. Although different levels of field resistance to citrus canker have been reported in sweet oranges, they are usually not sufficient to provide adequate control of the disease. Ectopic over-expression of antibacterial genes is one of the potential strategies to increase plant resistance to bacterial diseases. Previous in vitro results showed that sarcotoxin IA, an antimicrobial peptide isolated from the flesh fly (Sarcophaga peregrina), can be efficient to control different plant pathogenic bacteria, including Xcc. Transgenic “Pera” sweet orange (Citrus sinensis [L.] Osbeck) plants constitutively expressing the sarcotoxin IA peptide fused to the PR1a signal peptide from Nicotiana tabacum for secretion in the intercellular space were obtained by Agrobacterium-mediated transformation using thin sections of mature explants. Citrus canker resistance evaluation in leaves of transgenic and non-transgenic plants was performed through inoculations with Xcc by infiltration and spraying. The Xcc population was up to 2 log unit lower in leaves of the transgenic plants compared to those of non-transgenic controls. Incidence of canker lesions was significantly higher in non-transformed controls (>10 lesions/cm²) than in the transgenic plants (<5 lesions/cm²) after injection infiltration or spraying with Xcc inoculum. Accumulation of sarcotoxin IA peptide in sweet orange tissue did not cause any deleterious effects on the growth and development of the transgenic plants, indicating this approach is suitable to provide resistance to citrus canker.     

Scientific critique of the paper “Climatic distribution of citrus black spot caused by <Emphasis Type="Italic">Phyllosticta citricarpa</Emphasis>. A historical analysis of disease spread in South Africa” by Martínez-Minaya et al. (2015)

The global distribution of citrus black spot (CBS) disease, caused by Phyllosticta citricarpa, is climatically constrained, which is evident from its occurrence in citrus growing areas with warm, summer rainfall and its absence from areas with cooler, Mediterranean-type winter rainfall. Various epidemiological and modelling studies have supported this observation, predominantly estimating unsuitability for P. citricarpa in Mediterranean type climates, with no more than marginal suitability estimated at a few localities within some regions with Mediterranean type climates. The study by Martínez-Minaya et al. (European Journal of Plant Pathology, 143, 69–83, 2015), describes an historic sequence of recorded CBS occurrence in parts of South Africa, conducts an autocorrelation analysis and a correlative analysis with Köppen-Geiger climate zones and makes observations about the occurrence of certain Köppen-Geiger climate zones in the European Union. The study suggests that significant portions of the European Union and the broader Mediterranean basin are climatically similar to warm, summer rainfall areas in South Africa where P. citricarpa persists and causes CBS disease and concludes that the potential distribution of P. citricarpa is less constrained by climatic factors than spatial contagion. However, in this critique we expose methodological shortcomings in the Martínez-Minaya et al. (European Journal of Plant Pathology, 143, 69–83, 2015) study and conclude that the study grossly overestimated the extent of the geographical area that could support P. citricarpa, thereby rendering the findings scientifically unreliable.     

Histopathology of black spot symptoms in sweet orange<Emphasis Type="Italic">s</Emphasis>

In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immuno-histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols.     

Postharvest anthracnose on Satsuma mandarin orange caused by <Emphasis Type="Italic">Colletotrichum fioriniae</Emphasis>

In early January 2015–2017, anthracnose was detected on Satsuma mandarin orange (SMO) (Citrus unshiu) fruits kept in farmers’ storage rooms in Saga Prefecture, Japan. Three single-spore isolates from rotten fruits reproduced the postharvest anthracnose symptoms in wound-inoculated SMO fruits and were re-isolated from lesions. The isolates were identified as Colletotrichum fioriniae based on conidial morphology, culture characteristics, rDNA-ITS, and beta-tubulin-2 gene sequences. This is the first report of postharvest anthracnose on SMO caused by C. fioriniae.     

Citrus black spot severity related to premature fruit drop in sweet orange orchards

Citrus black spot (CBS) may cause substantial yield loss in sweet orange orchards. Severity and distribution of CBS symptoms on fruit may be variable and not all affected fruit may fall. However, the relationship between CBS symptoms and yield loss has not yet been investigated. This study aimed to determine CBS characteristics associated with premature fruit drop. A set of fruit was removed from the tree to measure the detachment force and another set of dropped fruit was collected from Pera and Valencia sweet orange orchards in three municipalities in Sao Paulo state, Brazil. CBS severity, distance of the nearest lesion from the peduncle, and fruit peel colour were assessed in both fruit sets. CBS severity was consistently related with the force to remove fruit and hence associated to the premature fruit drop, irrespective of the cultivar and location. The segmented linear regression showed that the fruit detachment force was reduced abruptly, at a rate of 7% for every 1% increase in disease severity up to 4.2 ± 1.0% CBS severity and, above this breakpoint, the force decreased slightly at a rate of 1.5%. Taking into account all orchards, the proportion of dropped fruit with CBS severity higher than the breakpoint was approximately 90%. This study provides a better understanding of the relationship between CBS severity and premature fruit drop of sweet orange. CBS severity breakpoint may be used as a reference to anticipate harvest and reduce CBS-related yield loss in orchards intended for juice processing.     

Identification of pathogenic fungi and preliminary screening for resistance in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. germplasm

Sexual reproduction in fungi is controlled by mating type genes, which are located at the MAT locus. In this study, we investigated the structure of this locus in the phytopathogenic fungus Phyllosticta citricarpa, the causal agent of citrus black spot disease. Despite intensive study, its sexual state has never been observed in single-spore culture. Through analysis of the genome sequences of two individual P. citricarpa isolates, the sequence of the DNA lyase gene was identified and, as previously reported in the literature, the mating type genes were located in the 3′ flanking region of this gene. The results suggested that P. citricarpa is heterothallic, owing to the exclusive presence of the MAT1–1 or MAT1–2 gene in individual strains. In order to characterize the MAT locus, we designed primers to amplify this region. P. citricarpa was found to have complete and apparently functional copies of MAT genes, containing α-1 and HMG domains, present in different isolates. In addition to MAT1–2-1 and MAT1–1-1 genes, the MAT1–1-4 gene was located in the 5′ flanking region of the MAT1–1-1 gene and the MAT1–2-5 gene was located in 5′ flanking region of the MAT1–2-1 gene. A multiplex PCR protocol was also developed to differentiate P. citricarpa idiomorphs, which can be used in distribution and incidence studies of mating type strains, in order to determine the occurrence of sexual reproduction and to facility crossing studies. Furthermore, in Brazil, the two idiomorphs occur in a 1:1 ratio, which is expected in sexually reproducing populations.     

Species‐specific real‐time PCR for diagnosis of Phyllosticta citricarpa on Citrus species

Phyllosticta citricarpa (teleomorph Guignardia citricarpa) is the causal agent of citrus black spot, a disease causing lesions on fruits and leaves of different Citrus species in Asia, Australia, South Africa and South America. It is a quarantine organism in the European Union and the USA and hence a reliable differentiation between this species and other Phyllosticta species found on Citrus is essential. A differentiation based on morphology is often problematic, hence a range of molecular tests have been developed to distinguish P. citricarpa from other species present on citrus fruits, especially the endophyte Phyllosticta capitalensis (teleomorph Guignardia mangiferae). However, these tests cannot distinguish P. citricarpa from the closely related Phyllosticta citriasiana, the causal agent of tan spot disease. In this study, a real‐time PCR was designed which is specific for P. citricarpa and does not amplify P. citriasiana or P. capitalensis DNA.     

Potential distribution of citrus black spot in the United States based on climatic conditions

Citrus black spot (CBS), caused by Phyllosticta citricarpa McAlp Van der Aa, was recently detected in southern Florida in the US. In addition to infected plant propagation materials, movement of infected citrus fruit poses a concern for potential spread of the disease out of the current quarantine zone, because lesions with pycnidia and conidia could develop after harvest. The conditions conducive for mycelial growth and development of pycnidia and conidia are not well known. Therefore, effects of temperature and relative humidity on growth and conidial production of P. citricarpa were determined and used as parameter inputs in CLIMEX to predict potential establishment of CBS in North America. Colony growth and conidial production in vitro were optimal at 27 °C, whereas there was no growth below 4 °C and above 37 °C. On fruit, lesion development and conidial production were observed at 4 °C, though at a low rate, indicating a greater versatility of the fungus on fruit. More full pycnidia were produced on the CBS lesions at 91 % RH compared to 84 %. Input parameters for CBS risk in CLIMEX obtained from literature, which reflected conditions for infection in spring/summer in Florida, predicted potential establishment in Florida but not in California. However, altering the parameter values to account for survival of the pathogen in leaf litter in winter predicted potential establishment in California as well as Florida. Thus, P. citricarpa could possibly establish beyond Florida if this organism is transported outside of the current quarantine zone to other citrus production areas.     

<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp. <Emphasis Type="Italic">michiganens</Emphasis>is strains virulence and genetic diversity. a first study in Argentina

Tomato leaves showing severe leaf spot symptoms have been observed and sampled in the central west and southwest Taiwan during 2015 and 2016. The symptoms were similar to those of bacterial leaf spot/late blight diseases, but only Stemphylium-like fungi were consistently isolated from the diseased tomato. Upon spray inoculation of tomato, Stemphylium-like isolates caused leaf spot symptoms identical to those of naturally infected plants, and the pathogenic isolates were successfully re-isolated from inoculated leaves. The tomato-pathogenic isolates were identified as S. lycopersici based on morphological characterization and molecular identification. S. lycopersici has been previously reported to cause gray leaf spot of tomato in the temperate regions, but the majority of S. lycopersici-caused lesions were black/dark brown rather than gray in our surveillance. Accordingly, it is suggested that S. lycopersici-caused disease of tomato is named Stemphylium leaf spot of tomato more appropriately than tomato gray leaf spot. Moreover, S. lycopersici-caused leaf spot disease on tomato has been distributed in major tomato production regions in Taiwan. The information provided by our study will be important for future breeding of tomato cultivars, especially for tomato producers in Taiwan.     

First report of flyspeck of mango caused by <Emphasis Type="Italic">Stomiopeltis</Emphasis> sp. in Japan

Flyspeck symptoms were found on mango (Mangifera indica L.) in Okinawa Prefecture, Japan in June 2014. Just before harvest, surfaces of fruits and green branches developed groups of tiny black dots within dark smudges. A fungus was isolated from the dots on both fruits and branches. The isolates were identified as Stomiopeltis sp. based on pathogenicity, morphology and molecular phylogenetic analyses with rDNA-ITS and LSU sequences. This is the first report of flyspeck (susuten-byo in Japanese) on mango caused by Stomiopeltis sp. in Japan.     

Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker

Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neo-nicotinoid that breaks down in planta into 6-chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1–3 log units in soil-treated plants compared to inoculated untreated plants.     

Reclassification of an isolate of Guignardia citricarpa from New Zealand as Guignardia mangiferae by sequence analysis

Citrus black spot was recorded as present in New Zealand in international databases on the basis of one isolate (ICMP 8336) identified by morphological features as Guignardia citricarpa . This isolate was from a soft rot, not a typical symptom of citrus black spot, on a Seminole tangerine fruit grown in the northern region of New Zealand. Sequence analysis of the internal transcribed spacer (ITS) region (ITS1, 5·8S and ITS2) showed that this isolate was 99% identical to the ITS region of G. mangiferae , a closely related saprotroph. Despite climatic conditions being suitable for this disease, citrus black spot symptoms have never been seen on citrus fruits grown in New Zealand. Thus the absence of symptoms on citrus in New Zealand is probably because G. citricarpa is not present. On the basis of these results, the record of citrus black spot occurring in New Zealand should be re-examined.     

Scytalidium Wilt of Citrus

In July 1998, a sudden wilt of Star Ruby grapefruit (Citrus paradisi Macf.) occurred in Israel in a region with a warm Mediterranean climate. The wilt of the middle and upper canopy of main limbs was accompanied by gum oozing from the affected branches. The bark of these branches attained a dark colour and the epidermis sloughed off easily revealing a mass of black powder, resulting from copious sporulation of dark conidia. Both the bark and the xylem were intensively colonized with mycelium. The fungus was identified as Scytalidium lignicola Pesante, based on the characteristic mixture of some colourless and dark conidia, produced in branched chains by conversion of the vegetative hyphae. Artificial inoculations induced typical disease symptoms and the fungus was reisolated, thus confirming Koch's postulates. Similar symptoms appeared in Star Ruby interim segments which had been cut and top grafted with various citrus cultivars. This is the first detailed report of pathogenicity of S. lignicola to citrus trees. It is postulated that the disease developed after predisposition of Star Ruby grapefruit trees by comprehensive pruning followed by extremely hot conditions.     

Control of infection of tomato fruits by <Emphasis Type="Italic">Alternaria</Emphasis> and mycotoxin production using plant extracts

Tomato fruits are susceptible to infection by Alternaria species. In addition, Alternaria species may contaminate the fruits with mycotoxins. There is thus interest in control systems to minimise pathogenicity and control toxin production. The objectives of this study were to examine the effect of plant extracts of Eucalyptus globulus and Calendula officinalis on the growth of strains of Alternaria alternata and A. arborescens, on pathogenicity of tomato fruits and mycotoxin production. The growth bioassays showed that the ethanolic and chloroformic fractions of E. globulus were the most effective in reducing growth of A. alternata (66–74 %) and A. arborescens (86–88 %), respectively at 2500 μg/g. The effects of plant extracts on mycotoxin biosynthesis were variable and strain dependent. The most effective fractions in decreasing mycotoxin accumulation were the ethanolic and chloroformic extracts of E. globulus, which reduced tenuazonic acid by 89 %, alternariol by 75–94 % and almost complete inhibition of alternariol monomethyl ether. All the tested fractions reduced percentage of infected tomato fruits when compared to the controls. The ethanolic and chloroformic fractions of E. globulus completely inhibited growth of A. alternata and A. arborescens on unwounded fruits and reduced the aggressiveness on wounded fruits of strains of both species significantly.     

Fruit spot of sweet lime (<Emphasis Type="Italic">Citrus limetta</Emphasis>) caused by <Emphasis Type="Italic">Septoria</Emphasis> sp. in Peru

In 2002, a severe fruit spot of sweet lime (Citrus limetta) was observed in Piura and Lambayeque provinces in northern Peru. Affected fruits showed large oval and sunken lesions, often surrounded by chlorotic haloes. Septoria sp. was isolated from affected fruits. Sweet lime isolates showed larger pycnidia and pycnidiospores than those of Septoria spp. previously described on citrus. In addition, phylogenetic analysis of the ITS sequences clearly separated the sweet lime isolates from S. citri and S. citricola. Isolates were pathogenic to detached sweet lime fruits and the fungus was isolated from lesions on inoculated fruits.     

Characterization and pathogenicity of <Emphasis Type="Italic">Bipolaris peregianensis</Emphasis>: the causal organism for leaf spot of hybrid bermudagrass in China

Several golf courses established with hybrid bermudagrass (Cynodon dactylon × C. transvaalensis) were surveyed from April 2011 through April 2015 in Hainan Province, China. The hybrid bermudagrass in these golf courses showed a new leaf spot disease, and a filamentous fungus was consistently recovered from the infected leaves. Based on the morphological characteristics of colony color and appearance, shapes of conidiophores and conidia as well as sequences of internal transcribed spacer regions (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the fungus was identified as Bipolaris peregianensis. The pathogenicity test conducted on healthy hybrid bermudagrass produced leaf spot symptoms one week post inoculation. B. peregianensis mycelia grew in a temperature range of 5 to 35 °C with the optimum temperature being 28 °C.