Integrated Management Approach to Citrus Fungal Diseases by Optimizing Cocoa-Based Agroforests Structural Characteristics

The health and productivity of citrus are generally jeopardized by a host of diseases, for which the environmental conditions of the cropping system are critical drivers. Several studies conducted on various diseases of perennial crops have shown the involvement of the structural futures of the cocoa-based agroforestry system (CBAFS) in the spread of pathogens and the epidemics development. This chapter highlights the effect of the CBAFS’s structural characteristics on the intensity of three citrus diseases in the humid forest zones of Cameroon. The involvement of CBAFS structural characteristics in diseases regulation is demonstrated. In particular, the spatial structure of citrus in agroforests shows an effect on the spread of diseases. Moreover, distribution of citrus in the CBAFS, with minimum spacing of 12 m between citrus trees, limits the damage caused by Pseudocercospora leaf and fruit spot disease (PLFSD) and citrus diseases caused by Phytophthora (CDP). Dense shading helps to minimize the intensity of diseases such as CDP and PLFSD and Citrus scab disease. This work may make it possible to contribute to the development of an integrated management tool for citrus diseases in an associated crop context.

First Report of Xanthomonas arboricola pv. pruni Causing Leaf and Fruit Spot on Apricot (Prunus armeniaca L.) in Montenegro

In July 2020, symptoms of leaf and fruit spot were observed on two-year old apricot plants (Prunus armeniaca L.), cultivar Rubista in plantation covering approximately 0,5 ha near Podgorica, central Montenegro. The intensity of infection on leaves was more than 70%. Initially, leaf spots were mainly circular, 2 to 5 mm in diameter, water-soaked, surrounded by a weak chlorotic halo, but later became light to dark brown and necrotic. Eventually, the spots merged and necrotic tissue dropped out, leaving a “shot-hole” leaf appearance. On apricot fruits small, dark brown, mainly circular superficial lesions were observed. The lesions merged and formed large necrotic areas reducing the quality of fruits. Symptoms were not observed on woody parts, such as twigs or stem. A total of 10 bacterial strains, forming yellow, convex, and mucoid colonies on yeast extract–dextrose–CaCO3 (YDC) medium, were isolated from symptomatic leaf and fruit tissue. All strains induced hypersensitive reaction in tobacco leaves. They were Gram-negative, strictly aerobic, oxidase negative, catalase positive, hydrolyzed gelatine and esculin but not starch, and did not grow at 37°C, showing similar biochemical properties as a reference strain Xanthomonas arboricola pv. pruni (Xap) (NCPPB 416) used in all tests as a positive control. Strains were further identified by PCR analysis, using primer pair XapY17-F/XapY17-R (Pagani 2004; Pothier et al. 2011), resulting in a single band of 943 bp, characteristic for Xap. Additionally, BOX-PCR with the BOX A1R primer (Schaad et al. 2001) showed 100% homology in genetic profiles of all tested strains and control strain. Amplification and partial sequencing of the gyrB gene of four representative strains was performed using set of primers described by Parkinson et al. (2007). Obtained DNA sequences showed that analysed strains (GenBank nos. MW473770, MW473771, MW473772, and MW473773) share 99.44 to 99.57% of gyrB sequence identity with Xap pathotype strain ICMP51. Pathogenicity of all strains was confirmed by spraying young apricot shoots using a hand-held sprayer, and by infiltration of apricot leaves (cv. Roksana) from the abaxial surface using a syringe without needle, with the bacterial suspension (107 CFU/ml in sterile distilled water), in three replicates. Sterile distilled water and reference Xap strain (NCPPB 416), were used as negative and positive controls, respectively. The inoculated shoots and leaves were maintained at approx. 25°C and high humidity conditions. Tissue necrosis appeared on all inoculated shoots 5 to 11 days and leaves 5 to 9 days after inoculation. Koch’s postulates were completed by re-isolation of the pathogen from inoculated tissue and identification by PCR using XapY17-F/XapY17-R primers. Based on pathogenic, biochemical and molecular characteristics, the strains isolated from apricot leaves and fruits in Montenegro were identified as Xap - causal agent of bacterial leaf spot and canker of stone fruits. This quarantine pathogen was previously reported on almond (Panić et al. 1998) and on peach (Popović et al. 2020) in Montenegro. This is the first report of Xap affecting apricot in this country. Therefore, strict phytosanitary measures have to be implemented to prevent spread of the pathogen in other areas and other susceptible hosts.

Development of loop-mediated isothermal amplification (LAMP) assay for detection of Pseudocercospora angolensis in sweet orange

Pseudocercospora angolensis is the causative agent of Pseudocercospora leaf and fruit spot disease in citrus which can result in up to 100% yield loss. Early diagnosis of this disease is vital for effective control. This study aimed at developing a loop-mediated amplification (LAMP) system for detecting P. angolensis in sweet oranges in comparison with Polymerase Chain Reaction (PCR) and using microscopy as a gold standard. Twelve non-target species were used to assess the analytical specificity of LAMP and PCR whereas the analytical sensitivity was determined using serial dilutions of P. angolensis DNA. The diagnostic accuracies of the two assays were evaluated using DNA from 150 diseased and 50 non-diseased sweet orange leaf samples. The analytical sensitivity and detection time of LAMP were of 10−4 ng/ μl and 40 minutes, respectively. The analytical sensitivity of PCR was 10ng/μl and it was specific to P. angolensis whereas three relatives of P. angolensis were detectable by LAMP. The diagnostic sensitivities of LAMP (93%) and microscopy (100%) were significantly different (X2 = 8.38, P = 0.0038) unlike the diagnostic specificities (90%) and (100%), respectively (X2 = 3.37, P = 0.066). Microscopy was significantly more sensitive than PCR (32.6%) (X2 = 149.26, P < 2.2e-16) and equally specific as PCR (P=NA). The positive predictive values of PCR and LAMP were 100% and 96.5% respectively whereas the negative predictive values were 33.1% and 81.8% respectively. The LAMP assay developed in this study offers a great tool for routine screening sweet orange samples for P. angolensis.

Correction to: Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach

Authors would like to update the given name and family name of authors which were incorrect in the original version.

Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach

The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).