Following the effects of micronutrient supply in HLB-infected trees: plant responses and ‘Candidatus Liberibacter asiaticus’ acquisition by the Asian citrus psyllid

Abstract

Despite efforts of research to establish best nutrient management practices in HLB-affected citrus orchards, there are still doubts about the contribution of such strategies to minimizing losses caused by the disease in the citrus industry. We evaluated the effects of micronutrient (Zn, Mn, and/or Cu) supply and 'Candidatus Liberibacter asiaticus' (CLas) infection on physiological and growth traits of sweet orange trees (Citrus sinensis (L.) Osbeck)) to understand if enhanced micronutrient supply would improve the growth of plants infected with CLas and reduce bacterial infection, as well as the acquisition of CLas by adults and nymphs of Diaphorina citri, correlated with nutritional status of trees. Plants were either grafted with buds obtained from micrografted plants (healthy, −) or buds infected with CLas (diseased plants, +). Infected plants were exposed to one of the five nutrient treatments, applied to leaf and root: (i) nilZnMnCu+: not fertilized with Cu, Mn, and Zn; (ii) Zn+: fertilized with zinc sulfate (ZnSO 4 .H 2 O); (iii) Mn+: fertilized with manganese sulfate (MnSO 4 .H 2 O); (iv) Cu+: fertilized with copper hydroxide [Cu(OH) 2 ]; and (v) ZnMnCu+: fertilized with all three micronutrients. Likewise, healthy plants were exposed to one of the two treatments as above: (i) nilZnMnCu− or (v) ZnMnCu−. We found that CLas impairs plant biomass production regardless of nutrient treatments, especially root growth, and increases specific leaf dry weight as disease progresses because of starch accumulation. Moreover, individual supply with Zn, Mn, or Cu can mitigate such deleterious effects of HLB on starch metabolism. HLB also changes nutrient concentrations in both leaves and sap extract, regardless of nutrient treatments, although treatments do not reduce the CLas titer in plants as determined by RT-qPCR. On the contrary, micronutrients applied in combination (ZnMnCu+) can reduce the acquisition of CLas by adults and especially nymphs of D. citri, likely reducing the disease infection in citrus orchards.

Sap Analysis: A Powerful Tool for Monitoring Plant Nutrition

“…We propose collecting 30 to 60 whole citrus leaves (including petioles) for extracting enough sap for one sample-suggesting no more than three leaves per tree-and a separate analysis of each sample. This coincides with the methodology used by [57] and [58], who took 40 leaves when working with sweet orange (Citrus sinensis) cultivars.…”

“…Instead of using petioles, in [55] and [56], the leaf blade midribs were used for sap analysis in broccoli (Brassica oleracea) and sugar cane (Saccharum officinarum), respectively, while in [52], the use of leaf blade vs. petioles was compared for sap analysis in strawberries (Fragaria × ananassa). In [57][58][59], leaf blades for sap analysis was recommended, which is becoming an interesting adaptation of the method by private companies in the Netherlands [19].…”

“…A new leaf would be fully expanded, but from the latest flush, located close to the growing point and with a light green color. Few authors have followed this approach of collecting old and new growth [58]. Most of the published work related to sap analysis has been focused on N and mostly in greenhouse crops, taking the most recent fully expanded leaves and petioles [2,19,28,29,39,40,53,55,56,60].…”

“…Later, the sample is defrosted, and the ether and chlorophyll solution (green colored fluid) is separated from the sap by a funnel. This methodology was followed by [27,30,[56][57][58]. Some authors also froze and defrosted tissues before pressing, but they did not mention the use of ether in their methods [28,53].…”

Interactions between Indigenous Endophyte Bacillus subtilis L1-21 and Nutrients inside Citrus in Reducing Huanglongbing Pathogen Candidatus Liberibacter Asiaticus

“…Zambon et al [46] reported beneficial effects of Mn foliar application with three times higher than the recommended dose in HLB-affected trees have shown reduction of CLas titer and improvement of fruit production [46,47]. Therefore, it can be proven that nutrients have great importance in the interaction of the plant host, microbial community and vectors [14,46,48]. Most of essential nutrients influenced the severity of plant disease [49].…”