Efficacy of Trichoderma-based formulates against Mal secco disease of citrus

Species distribution models are widely used to estimate the potential geographic distribution of species habitat. They are also used to predict the impact of climate change on species distributions. In this study, we examined the distribution of Mal Secco disease, an infective disease of citrus caused by the fungus Plenodomus tracheiphilus . To model the Mal Secco distribution under current and two future (2050 and 2070) climatic scenarios in the Mediterranean basin, eight climate variables were incorporated into the Maximum Entropy (MaxEnt) model. All three model distributions had high AUC values (0.97), indicating excellent performance. The precipitation during the wettest month and the minimum temperature during the coldest month contributed the most to the model. The suitable areas for the Mal Secco disease were predicted to decrease by up to 23% by the year 2070. For each of the three scenarios, the change in the proportion of suitable areas along the longitudinal (west–east) and latitudinal (south–north) geographical axes of the Mediterranean basin showed a similar trend. Our study highlights the supposition that while climate change is likely to reduce the extent of suitable areas, no range shift is expected to occur in the Mediterranean basin.

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.

Mal secco, caused by Plenodomus tracheiphilus, is an economically important fungal vascular disease in citrus-growing countries of the Mediterranean basin. Preventing fungal infections usually requires a high number of copper treatments but European legislation imposes the minimization of their accumulation in soil. In our study, biological control agents (BCAs) and a plant resistance inducer (PRI), tested in four different experiments on citrus seedlings under controlled conditions, have resulted in promising strategies to control mal secco disease. Foliar (Experiment I) and soil (Experiment II) applications of two formulations of Bacillus amyloliquefaciens strain D747 (Amylo-X® LC and Amylo-X® WG) provided similar performances in reducing the disease amount (incidence and symptoms severity) over time compared to the untreated control, whereas copper hydroxide (Kocide Opti®) used as standard was the most effective treatment over time. In the third experiment, Pythium oligandrum strain M1 (Polyversum®) and Trichoderma asperellum strain ICC012 + Trichoderma gamsii strain ICC080 (Remedier®) were able to reduce disease incidence and symptoms severity compared to the untreated control. Remedier® provided the best performances in reducing the disease amount, whereas the Polyversum® application was the least effective treatment over time. The effectiveness of the Trichoderma spp. formulation in reducing P. tracheiphilus infections did not significantly differ from the standard copper compound (Kocide Opti®). Comprehensively, in the last experiment (IV), acibenzolar-S-methyl (ASM) alone and in mixture with metalaxyl-M proved as effective as B. amyloliquefaciens strain FZB24, with no dose–response relationships observed. These findings provide important insight for the integrated management of mal secco disease.

The effectiveness of biological commercial products based on Bacillus amyloliquefaciens strains was evaluated through in vitro and in vivo experiments against Plenodomus tracheiphilus. The activity of bacterial cells, volatile organic compounds (VOCs), and culture filtrates of bacteria were tested in vitro against different isolates of P. tracheiphilus. Afterwards, the virulence of these isolates was evaluated on Citrus volkameriana plants to select the most virulent isolate to use in the in vivo experiments. To evaluate the effectiveness of products, C. volkameriana seedlings were pre-treated, twice with biological products and once with standard fungicides, before pathogen inoculation. Moreover, in order to determine the endophytic ability of the bacteria, the population density within the treated citrus stem was determined. Comprehensively, bacterial cells, filtrates, and VOCs were able to significantly reduce the average mycelial diameter of P. tracheiphilus, with some variability according to pathogen isolate. In planta experiments showed that the biological products on average were less effective than fungicides, although all formulates were able to significantly reduce disease incidence and symptom severity, except B. amyloliquefaciens strain D747 (Amylo-X) for symptom severity (SS) 20 days after inoculation. Bacteria were re-isolated from the internal woody tissue of treated plants, showing strong endophytic ability. This work is important as commercial biological products based on B. amyloliquefaciens strains could represent a promising and sustainable alternative for the integrated management of mal secco disease.

Mal secco disease of citrus is caused by the mitosporic ascomycete fungus Plenodomus tracheiphilus (formerly Phoma tracheiphila [Petri]). Mal secco is a highly destructive vascular disease of lemon and other citrus which is presently confined to the Mediterranean basin and has a marked economic impact on the citrus industry. The fungal pathogen infects the host tree by penetrating through wounds in the roots or canopy. Infection spreads quickly into the main branches and trunk and tree mortality usually ensues. The most typical symptoms are veinal chlorosis, leaf wilt, red discoloration of the xylem and dieback of twigs and branches. Current accepted control of the disease is mainly by sanitation of infected wood, and copper application during the winter to prevent germinating spores from infecting the plant. No effective chemical control for this disease has been reported. We present a drip-irrigation protocol to protect trees and control the disease in which we apply 250 g/ha of the triazole fungicide flutriafol five times a year. Progression of disease symptoms in the treated trees was inhibited by up to 81% compared to the untreated control, thereby significantly shortening the sanitation process and making it less costly than in untreated trees. Moreover, disease symptoms became less severe as the duration of treatment increased. We have treated orchards for 3 years, and present an effective commercial protocol for the growers which will help them control Mal secco disease.

The lemon industry in the Mediterranean basin is strongly threatened by "mal secco" disease (MSD) caused by the fungus Plenodomus tracheiphlilus. Leaf pretreatments with Pseudomonas mediterranea 3C have been proposed as innovative tools for eco-sustainable interventions aimed at controlling the disease. In this study, by exploiting the results of previously performed RNAseq analysis, WCGNA was conducted among gene expression patterns in both inoculated (Pt) and pretreated and fungus-inoculated lemon plants (Citrus limon L.) (3CPt), and two indicators of fungal infection, i.e., the amount of fungus DNA measured in planta and the disease index (DI). The aims of this work were (a) to identify gene modules significantly associated with those traits, (b) to construct co-expression networks related to mal secco disease; (c) to define the effect and action mechanisms of P. mediterranea by comparing the networks. The results led to the identification of nine hub genes in the networks, with three of them belonging to receptor-like kinases (RLK), such as HERK1, CLAVATA1 and LRR, which play crucial roles in plant-pathogen interaction. Moreover, the comparison between networks indicated that the expression of those receptors is not induced in the presence of P. mediterranea, suggesting how powerful WCGNA is in discovering crucial genes that must undergo further investigation and be eventually knocked out.

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.

“Mal secco”, an Italian name meaning “dry disease”, is a severe tracheomycotic disease of citrus caused by the mitosporic fungus Phoma tracheiphila (Petri) Kantsch. et Gik. It appeared in 1894 in two Aegean Greek islands, from which it spread almost to the whole Mediterranean basin and the Black Sea. Due to its high susceptibility, lemon is the most damaged citrus species. Disease damage consists of substantial reduction of the quality and quantity of the crop, mainly due to the difficulties of controlling the disease and the replacement of susceptible valuable cultivars by others which are less vulnerable, but have low productivity and scarce fruit quality. Control of mal secco disease has relied on a number of diverse chemical and nonchemical strategies, but is still faced with efficacy problems. Host resistance remains a most desirable goal, but it will not be ultimately achieved until the genetic basis of resistance to P. tracheiphila are not fully elucidated. The present paper reviews the different aspects of citrus mal secco as studied worldwide over almost a century of research, from the first appeareance of the disease in Italy (1918) to date. Milestones and pitfalls about the symptomatology, aetiology, host-parasite relationship, diagnosis, epidemiology, and control are discussed in a historical perspective, emphasizing the advancements in knowledge. Finally, some issues and challenges are highlighted that need to be more comprehensively addressed prior to deployment of effective disease control measures.

<i>Phoma tracheiphila</i>

A role for oxidative stress in the <i>Citrus limon</i>/<i>Phoma tracheiphila</i> interaction

Mycorrhizal fungi represent one of the oldest and most successful symbioses in plant evolution. Communication among mycorrhizal fungi and plants occurs prior to direct contact among them through different and variable biochemical signals, including microRNAs, hormones, small peptides and volatile organic and inorganic compounds. Volatile organic compounds (VOCs) emerge as key chemical signals that enable the transmission of chemical messages modulating plant and microorganism responses in both below- and above-ground ecosystems. The diversity and concentration of mycorrhizal VOCs will vary depending on the environment and the emitting organism and are usually related to changes in the conformation of root architecture and lateral root formation mediated by auxin and strigolactones. Moreover, the study of the effects of mycorrhizal VOCs in the tolerance to abiotic and biotic stress are still scarce although there are some promising results pointing out to the effect of these VOCs in plant development under osmotic stress conditions, and their properties as antifungal and antibacterial molecules. However, the information regarding the molecular mechanisms involved in mycorrhizal VOCs signaling and their effect on plants remains still elusive. The understanding of VOC-mediated plant-mycorrhizal interactions, together with the technical improvements for their detection and mode of application in the field, will open new avenues for biotechnological crop improvement and management that not only will reduce the dependence on agrochemicals but also fosters soil health and plant resilience.

Mal secco is a vascular disease of citrus caused by the mitosporic fungus Plenodomus tracheiphilus. Soil containing infected plant material constitutes an inoculum source for root infections. In this study, the soil bacterial and fungal communities of five lemon orchards located in Syracuse Province (Sicily, Italy) affected by mal secco were analyzed. Soil samples were collected under lemon tree canopies and subjected to total genomic DNA extraction. The fungal DNA was detected through qPCR in all orchards, with variable concentrations. Bacterial and fungal communities were profiled using 16S and ITS amplicon-based high-throughput sequencing, respectively. According to our results, the relative abundances of the most represented bacterial phyla (e.g., Proteobacteria, Actinobacteriota, Acidobacteriota) changed across the orchards, while in the fungal community, the phylum Ascomycota was dominant, with Basidiomycota and Mortierellomycota abundances fluctuating. On the whole, β diversity analysis showed significant variation in the composition of the soil microbial communities across the orchards. This result was confirmed by the analysis of the core community (taxa present at ≥ 75% of total samples), where putative beneficial bacteria resulted in significantly enriched fungus-infected soil samples, suggesting complex microbial interactions. Our findings shed light on the composition and diversity of the soil microbiome in lemon orchards with the occurrence of mal secco infections.

‘Mal secco’ caused by Phoma tracheiphila (Petri) Kanc. et Ghick. is a vascular disease of citrus occurring in the Mediterranean areas. Leaves and twigs suddenly wilt and dry, frequently in one section of the tree. Large branches and finally the whole tree may die back in few years. In culture two types of strains can be distinguished: chromogenic and non chromogenic (Baldacci, 1950). Several of the substances responsible for the chromogenic pigmentation were identified (Quilico et al., 1952; Balli o et al., 1979; Lerario et al., 1986). The present study was designed to determine the ability of the chromogenic strains to produce an-thraquinones on different media in vitro.

In this study, we investigated the biocontrol activity of the P. mediterranea strain PVCT 3C against Mal secco, a severe disease of citrus caused by the vascular fungus Plenodomus tracheiphilus. In vitro, bacterial diffusible compounds, volatile organic compounds and culture filtrates produced by PVCT 3C reduced the mycelial growth and conidial germination of P. tracheiphilus, also affecting the mycelial pigmentation. The application of bacterial suspensions by leaf-spraying before the inoculation with the pathogen on plants of the highly susceptible species sour orange and lemon led to an overall reduction in incidence and disease index, above all during the early disease stage. PVCT 3C genome was subjected to whole-genome shotgun sequencing to study the molecular mechanisms of action of this strain. In silico annotation of biosynthetic gene clusters for secondary metabolites revealed the presence of numerous clusters encoding antimicrobial compounds (e.g. cyclic lipopeptides, hydrogen cyanide, siderophores) and candidate novel products. During the asymptomatic disease phase (seven days post-inoculation), bacterial treatments interfered with the expression of different fungal genes, as assessed with an NGS and de novo assembly RNA-seq approach. These results suggest that P. mediterranea PVCT 3C or its secondary metabolites may offer a potential effective and sustainable alternative to contain P. tracheiphilus infections via integrated management.

The integration of management methods for both diseases and weeds depends on the compatibility between the tools. Biological control represents an important strategy to cope with the integrated management of white mold (Sclerotinia sclerotiorum) through parasitism of sclerotia. However, its application in the field is more cost-effective if combined with the herbicide in a tank mix, as long as the products are compatible. Therefore, we aimed at (i) evaluating two compatibility test methodologies (constant exposure and different times) and (ii) two soybean crop seasons to infer the compatibility of Trichoderma-based products. In vitro bioassays were performed to assess the compatibility between herbicides (Haloxifope-p- methyl, Glyphosate N-ammonium salt, Fluasifope-p-butyl, Fomesafem, Chlorimuron ethyl and Imazapyc + Imazapyr) and two biocontrol agents (Trichoderma asperellum and Trichoderma harzianum). Thus, the recommended spray volume for each herbicide was added to the PDA culture medium (Potato-Dextrose-Agar) and then deposited in the center of the plate a disc of mycelium from each antagonist isolate (constant exposure). The tests with time of exposure were marked at times 0, 2, 4, 8 and 16 h (simulating tank mixing) and at the spp of each time were plated on PDA medium. For both tests, the mycelial growth and conidiogenesis of Trichoderma spp. were observed. The combination of herbicide and biocontrol was also tested in the field and sclerotia parasitism, white mold incidence and plant yield were assessed in two field trials. The constant exposure of the antagonists to herbicides revealed that no herbicide was compatible with the T. asperellum or T. harzianum. While in test, exposure time exhibited compatibility with either T. asperellum or T. harzianum, within a period of 2 to 8 h. Conclusively, the integration of biocontrol agents with the herbicide imazapique + imazapyr exhibited significant reductions in white mold disease incidence and conidia germination, along with effective parasitism of S. sclerotiorum and even the least compatible herbicide (glyphosate) resulted in significant reduction in the disease incidence and sustained yield when compared to the untreated control. Therefore, the integration of the biocontrol agent for white mold should always be considered, and the tank mixing of it with the herbicide represents a cost-effective alternative for the grower.