Polarimetric imaging microscopy for advanced inspection of vegetal tissues

Optical microscopy techniques for plant inspection benefit from the fact that at least one of the multiple properties of light (intensity, phase, wavelength, polarization) may be modified by vegetal tissues. Paradoxically, polarimetric microscopy although being a mature technique in biophotonics, is not so commonly used in botany. Importantly, only specific polarimetric observables, as birefringence or dichroism, have some presence in botany studies, and other relevant metrics, as those based on depolarization, are underused. We present a versatile method, based on a representative selection of polarimetric observables, to obtain and to analyse images of plants which bring significant information about their structure and/or the spatial organization of their constituents (cells, organelles, among other structures). We provide a thorough analysis of polarimetric microscopy images of sections of plant leaves which are compared with those obtained by other commonly used microscopy techniques in plant biology. Our results show the interest of polarimetric microscopy for plant inspection, as it is non-destructive technique, highly competitive in economical and time consumption, and providing advantages compared to standard non-polarizing techniques.

Intraplant Distribution, Preferred Sites, and Damage Caused by Euschistus heros (Hemiptera: Pentatomidae) on Cotton

This study investigated (a) the intraplant distribution of Euschistus heros (F.) (Hemiptera: Pentatomidae) in cotton, (b) its preference for different structures and areas of the cotton plant, and (c) the damage caused at different levels of infestation. Our results determined that, regardless of the time of day, E. heros adults significantly preferred the middle third of cotton plants in comparison to the upper and lower areas (F = 30.221; df = 2; P = 0.001). The adults were present on all cotton structures, and numbers were significantly higher on bolls and leaves/branches (F = 25.333; df = 5; P = 0.001) than on flower buds and flowers. In greenhouse tests, fiber yield, number of bolls per plant, and number of internal punctures in bolls responded significantly to increasing infestation levels of E. heros adults. Damage and yield reduction was observed at 1.5 to 2.0 adults/plant, indicating that an action threshold of 1 adult per plant should be adopted to avoid economic losses. Sampling during boll development should be by either whole-plant inspection or beat cloth methods between 0600 and 1800 h.

Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing

‘Candidatus Liberibacter spp.’ are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of ‘Ca. L. asiaticus’ (CLas) in sweet orange. We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of ‘Ca. L. africanus’ (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.