Polyclonal Antibody-based ELISA in combination with specific PCR amplification of ITS 1 regions for the detection and quantitation of Lasiodiplodia theobromae, causal agent of 2 gummosis in cashew nut plants

Members of Botryosphaeriaceae family are associated with serious diseases in different plants 18 across the world. In cashew nut plants (Anacardium occidentale L.), the fungus Lasiodiplodia 19 theobromae causes a severe group of symptoms related to gummosis that results in decreased nut 20 production. The aim of this work was to develop an indirect enzyme-linked immunosorbent 21 assay (ELISA) with sufficient sensitivity and specificity to detect the fungus both in vitro and in 22 planta (artificially and naturally infected) and to increase the detection specificity within the 23 fungi group using primers specific for the internal transcribed spacer (ITS) sequences. A 24 collection of L. theobromae isolates was obtained, and antisera against the fungus were raised in 25 rabbits. Cross-reactivity against Neofusicoccum sp., Colletotrichum gloeosporioides, Phomopsis 26 anacardii and Pestalotiopsis guepinii was examined. Naturally and artificially infected vegetal 27 material was employed in the ELISAs. The fungi ITS sequences were determined, and single 28 nucleotide polymorphisms were identified and used for primer design. For the naturally infected 29 2 plants, there was an approximately 4-fold variation in the absorbance values. Some positive 1 readings for asymptomatic samples were detected. For the artificially infected samples, an 2 ELISA-based weekly time-course analysis was conducted, and the values for samples from 0 and 3 7 days were lower than the threshold value. Beginning on day 14, the infection could be 4 detected, with rates varying from 40% on day 14 to 80% on day 21 and 100% by the end of the 5 experiment. The ITS sequencing revealed few polymorphisms among the L. theobromae isolates, 6 but for Colletotrichum gloeosporioides, Phomopsis anacardii, Pestalotiopsis guepinii and 7 Neofusicoccum sp., the sequences were sufficient to permit reliable discrimination. The 8 feasibility of ELISA as an early detection technique to assist in gummosis management was 9 demonstrated. PCR amplification based on ITS regions increases and complements serological 10 specificit

Monitoring cashew seedlings during interactions with the fungus Lasiodiplodia theobromae using chlorophyll fluorescence imaging

The chlorophyll (Chl) fluorescence imaging technique was applied to cashew seedlings inoculated with the fungus Lasiodiplodia theobromae to assess any disturbances in the photosynthetic apparatus of the plants before the onset of visual symptoms. Two-month-old cashew plants were inoculated with mycelium of L. theobromae isolate Lt19 or Lt32. Dark-adapted and light-acclimated whole plants or previously labelled, single, mature leaf from each plant were evaluated weekly for Chl fluorescence parameters. From 21 to 28 days, inoculation with both isolates resulted in the significantly lower maximal photochemical quantum yield of PSII (Fv/Fm) than those for control samples, decreasing from values of 0.78 to 0.62. In contrast, the time response of the measured fluorescence transient curve from dark-acclimated plants increased in both whole plants and single mature leaves in inoculated plants compared with controls. The Fv/Fm images clearly exhibited photosynthetic perturbations 14 days after inoculation before any visual symptoms appeared. Additionally, decays in the effective quantum yield of PSII photochemistry and photochemical quenching coefficient were also observed over time. However, nonphotochemical quenching increased during the evaluation period. We conclude that Fv/Fm images are the effective way of detecting early metabolic perturbations in the photosynthetic apparatus of cashew seedlings caused by gummosis in both whole plants and single leaves and could be potentially employed in larger-scale screening systems

Processamento mínimo, atmosfera modificada, produtos químicos e resfriamento no controle da podridão basal pós-colheita em frutos do coqueiro anão verde Minimal processing, modified atmosphere, chemical products and cooling to control post-harvest basal rot of fresh green coconut fruits (Cocos nucifera)

O trabalho teve o objetivo de investigar, isolada e integradamente, o efeito do corte polar do coco verde, a atmosfera modificada, e a associação de produtos químicos, sob condições de câmara frigorífica no controle da podridão basal pós-colheita causada por Lasiodiplodia theobromae. Frutos provenientes de áreas infestadas foram cortados transversalmente, removendo-se as brácteas e, em seguida, foram submetidos aos tratamentos com ceras e fungicidas. Após o tratamento, esses frutos foram embalados em caixas de papelão e transferidos para a câmara frigorífica à ±12º C por 30 ou 35 dias, de acordo com o ensaio. As avaliações foram realizadas a cada dois dias. A exclusão do fungo por meio do corte do mesocarpo na região das brácteas do coco verde foi eficiente no controle da doença, e a proteção com a cera EF-1 foi excelente protetor contra o dano pelo frio e fungos deteriorantes nos frutos cortados. Também, verificou-se que a associação corte basal do fruto + emulsão de cera + fungicida foi uma excelente alternativa de conservação e controle da doença em estudo.<br>Fruits of coconut (Cocos nucifera) cultivated in Brazil for coconut milk consumption are highly susceptible to attack by the fungus Lasiodiplodia theobromae. Infection occurs still in the field as the pathogen remains quiescent in the plant. This study was undertaken to test several processing methods in order to avoid fruit deterioration. Fruits collected from a plantation with a record of severe disease outbreaks were cut at the basal region and treated with emulsions prepared from palm wax, alone and with fungicides. Fruits were cut 1.5 cm deep from the insertion point by using a cut machine developed for this task. fruits were placed in a paper box, and kept in a cool chamber at +12ºC for 30 to 35 days, depending upon the treatment. Evaluations were carried out every 2 days. The cutting process successfully eliminated the fungus from the fruit tissues. In addition, emulsion EF-1 exhibited an excellent protection against chilling damage as well as against opportunistic fungi. The association cut of basal section + emulsion + fungicide was the best alternative for controlling the disease