Phoma tracheiphila (Mal secco).

P. tracheiphila is a conidial fungus causing serious damage and death to the host, particularly lemon [Citrus limon], in Citrus and related genera, in Mediterranean countries and the Black Sea region. So far, however, it is unknown in Spain, Portugal and Morocco, as well as other major citrus-growing regions of the world. This fungus is of concern to many international plant protection organizations and must be prevented from being spread in infected propagative material. It was once reported as seedborne in lemon, but there is no further evidence of this means of transmission. Once in the orchard, the fungus can be carried as spores from pycnidia and from hyphae on the plant and on fallen debris by rain, wind and irrigation water, and perhaps by birds and insects.

Biotechnological Approaches for Genetic Improvement of Lemon (Citrus limon (L.) Burm. f.) against Mal Secco Disease

Among Citrus species, lemon is one of the most susceptible to mal secco disease, a tracheomycosis caused by the mitosporic fungus Plenodomus tracheiphilus, which induces chlorosis followed by leaf drop and progressive desiccation of twigs and branches. Severe infection can cause the death of the plant. Since no effective control strategies are available to efficiently control the pathogen spread, host tolerance is the most desirable goal in the struggle against mal secco disease. To date, both traditional breeding programs and biotechnological techniques were not efficient in developing novel varieties coupling tolerance to mal secco with optimal fruit quality. Furthermore, the genetic basis of host resistance has not been fully deciphered yet, hampering the set-up of marker-assisted selection (MAS) schemes. This paper provides an overview of the biotechnological approaches adopted so far for the selection of mal secco tolerant lemon varieties and emphasizes the promising contribution of marker-trait association analysis techniques for both unraveling the genetic determinism of the resistance to mal secco and detecting molecular markers that can be readily used for MAS. Such an approach has already proved its efficiency in several crops and could represent a valuable tool to select novel lemon varieties coupling superior fruit quality traits and resistance to mal secco.

De Novo Transcriptome Sequencing of Rough Lemon Leaves (Citrus jambhiri Lush.) in Response to Plenodomus tracheiphilus Infection

Mal secco is one of the most severe diseases of citrus, caused by the necrotrophic fungus Plenodomus tracheiphilus. With the main aim of identifying candidate genes involved in the response of citrus plants to “Mal secco”, we performed a de novo transcriptome analysis of rough lemon seedlings subjected to inoculation of P. tracheiphilus. The analysis of differential expressed genes (DEGs) highlighted a sharp response triggered by the pathogen as a total of 4986 significant DEGs (2865 genes up-regulated and 2121 down-regulated) have been revealed. The analysis of the most significantly enriched KEGG pathways indicated that a crucial role is played by genes involved in “Plant hormone signal transduction”, “Phenylpropanoid biosynthesis”, and “Carbon metabolism”. The main findings of this work are that under fungus challenge, the rough lemon genes involved both in the light harvesting and the photosynthetic electron flow were significantly down-regulated, thus probably inducing a shortage of energy for cellular functions. Moreover, the systemic acquired resistance (SAR) was activated through the induced salicylic acid cascade. Interestingly, RPM1 interacting protein 4, an essential positive regulator of plant defense, and BIR2, which is a negative regulator of basal level of immunity, have been identified thus representing useful targets for molecular breeding.

Identification of Field Tolerance and Resistance to Mal Secco Disease in a Citrus Germplasm Collection in Sicily

Mal secco is a tracheomycotic disease caused by the fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley that has caused severe damage and loss of yield in the citrus industry in the Mediterranean area, for 100 years. While the disease can affect different cultivated citrus species, lemon (C. × limon var. limon (L.) Burm. f.) and citron are the most susceptible. The identification of resistant or field-tolerant clones and hybrids is a major goal for lemon growers and breeders. To identify sources of resistance or tolerance to the disease, we performed a phenotypic survey on a lemon and lemon-like open-field germplasm planted at CREA (Research Centre for Olive, Fruit and Citrus Crops), Italy, in an area with high pathogen pressure. Phenotyping was performed visually, four times, for three consecutive years, on a total of 50 accessions, with two or three replicate trees per accession. Moreover, molecular screening based on real-time PCR was performed, for two consecutive years, on twigs, young leaves, and mature leaves of all plants, to detect the pathogen in the absence of clear symptoms. The accessions were categorized into seven groups based on the presence of visual symptoms, real-time PCR pathogen detection, and canopy volume. The results revealed sources of tolerance in lemon and citron hybrids. The molecular screening identified P. tracheiphilus in all lemon clones, with mean Ct values ranging from 17 to 39. The screening also identified P. tracheiphilus in clones without clear symptoms, indicating their ability to tolerate the disease. Moreover, a strong negative correlation was found between the Ct values in twigs and symptom severity (r = −0.72). This indicates that the DNA from twigs is the most appropriate for use in performing reliable phenotyping of mal secco susceptibility in adult plants. An autotetraploid lemon (Doppio Lentini) seems to be immune to the disease, under natural pressure, since P. tracheiphilus was not detected by real-time PCR and visual screening. Overall, the data obtained are a valuable resource for identifying both the most tolerant lemon varieties suitable for areas with high pathogen pressure and the best breeding parents for the introgression of resistance genes into lemon genotypes.

Spatial and Temporal Dynamics of Mal Secco Disease Spread in Lemon Orchards in Israel

Mal Secco is a severe disease of citrus in which the fungus Plenodomus tracheiphilus (formerly Phoma tracheiphila) penetrates the vascular system of the host. In this study, we characterized the spatial dynamics of the disease in seven lemon orchards. A representative block of trees from each orchard was evaluated monthly during 3 consecutive years. In addition, scouts assessed disease severity in 75 orchards from three different geographical regions and tested for association between disease severity and measures of orchard management, environmental factors, cultural practices, and cultivar type. We assessed disease incidence and characteristics of spatial patterns using Ripley’s K function and fitted logistic regression models for different neighboring tree structures followed by model selection methods to provide insight into the spatial and temporal dynamics of disease progress. We found different rates of disease spread in different orchards, which are most likely the result of differences in orchard management practices or less likely the result of differences in climatic conditions. There was an indication that agricultural tools contribute to spread of the disease within rows of trees. The results confirm that the lemon cultivar Interdonato is less susceptible compared with other citrus cultivars, and they suggest that the density of urban terrain surrounding each orchard is positively correlated with the severity of the disease. In contrast to our expectations, no correlation was found between the density of lemon orchards surrounding an orchard and the severity of the disease within it, which corroborates previous findings regarding the limited distribution of the disease.