MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss’s wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples

The Goss’s bacterial wilt pathogen, Clavibacter nebraskensis, of corn is a candidate A1 quarantine organism; and its recent re-emergence and spread in the USA and Canada is a potential biothreat to the crop. We developed and tested an amplicon-based Nanopore detection system for C. nebraskensis (Cn), targeting a purine permease gene. The sensitivity (1 pg) of this system in mock bacterial communities (MBCs) spiked with serially diluted DNA of C. nebraskensis NCPPB 2581T is comparable to that of real-time PCR. Average Nanopore reads increased exponentially from 125 (1pg) to about 6000 reads (1000 pg) after a 3-hr run-time, with 99.0% of the reads accurately assigned to C. nebraskensis. Three run-times were used to process control MBCs, Cn-spiked MBCs, diseased and healthy leaf samples. The mean Nanopore reads doubled as the run-time is increased from 3 to 6 hrs while from 6 to 12 hrs, a 20% increment was recorded in all treatments. Cn-spiked MBCs and diseased corn leaf samples averaged read counts of 5,100, 11,000 and 14,000 for the respective run-times, with 99.8% of the reads taxonomically identified as C. nebraskensis. The control MBCs and healthy leaf samples had 47 and 14 Nanopore reads, respectively. 16S rRNA bacteriomic profiles showed that Sphingomonas (22.7%) and Clavibacter (21.2%) were dominant in diseased samples while Pseudomonas had only 3.5% relative abundance. In non-symptomatic leaf samples, however, Pseudomonas (20.0%) was dominant with Clavibacter at 0.08% relative abundance. This discrepancy in Pseudomonas abundance in the samples was corroborated by qPCR using EvaGreen chemistry. Our work outlines a new useful tool for diagnosis of the Goss’s bacterial wilt disease; and provides the first insight on Pseudomonas community dynamics in necrotic leaf lesions.

FOLIAR BIOCHEMICAL CHANGES DURING GALL FORMATION IN MITRAGYNA PARVIFOLIA

Mitragyna parvifolia is a medicinally important plant which is native to India and Shrilanka. This plant is widely used by tribal people of Rajasthan and other Ayurvedic practitioners. Normal leaf and leaf gall of Mitragyna parvifolia were collected and categorized in normal (healthy leaf) and galled leaf. In the present investigation an attempt was made to study the changes of some metabolites during the gall formation on leaf of Mitragyna parvifolia attacked by gall insect. Total soluble sugar, total phenol and peroxidase activities were found high in gall leaves as compared to healthy leaves but chlorophyll contents and polyphenol oxidase activities were recorded high in normal leave and protein contents were slightly high in normal leaves as compared to infected or gall leaves.

Surgery or surgical defolation in ‘Grand Naine’ banana in the control of black Sigatoka in the state of Rio de Janeiro

‘Grande Naine’ banana (Musa acuminata - AAA) in the municipality of Angra dos Reis (RJ) has been affected by the Black Sigatoka agent, fijiensis Mycosphaerella(current name Pseudocercospora fijiensis); causing losses to local producers, mostly small producers and low-income producers. Since Black Sigatoka was reported in the mentioned municipality, it was aimed to determine control efficiency through surgical measurement or surgical defoliation and to evaluate its effect on banana productivity characteristics. For this purpose, treatment was designed in the form of completely randomized blocks containing three blocks, each block with five plants with treatment (Surgery) and five plants without treatment (Control), in a total of 30 plants with fungal infection. Treatments were performed every 15 days with monthly severity assessments (Weighted Mean of Infection - MPI) by the method of Stover modified by Gauhl. The results obtained at the end of the trial showed significant differences in all variables (Severity, youngest leaf spotted, bunch weight and fruit size), and treatment with surgery provided reduction in severity (MPI) around 58%, thus confirming that plants received adequate treatment according to the results obtained for this methodology, and, contributing to the increase of the healthy leaf area in the youngest leaves, whereas in plants without surgery (Control), severity increased by 14.8% in comparison to the initial MPI evaluation, consequently having a smaller healthy leaf area for the accomplishment of photosynthesis and other physiological processes. In production variables bunch weight, fruit size and number of fruits, differences were significant (Pr> F = 0.0001), reaching an increase in yield of up to 300% when compared to Control. It is therefore an effective method for the control of phytopathogen black sigatoka unpublished in the state of Rio de Janeiro and that will bring direct benefits for Family Agriculture in the region of incidence of this banana disease.