Four glutathione S-transferase (GST) genes, NbGSTU1, NbGSTU2, NbGSTU3, and NbGSTF1, were amplified from cDNA of Nicotiana benthamiana leaves infected with Colletotrichum destructivum using primers based on conserved regions of N. tabacum GST sequences. Expression of NbGSTU1 and NbGSTU3 increased progressively during infection by either C. destructivum or Colletotrichum orbiculare, except for a slight decrease by NbGSTU1 late in the infection, whereas NbGSTU2 and NbGSTF1 expression remained relatively constant. Each of the four genes was cloned into a PVX vector for virus-induced gene silencing, and reduced expression of the four genes was detected by RT-PCR. A statistically significant increase in susceptibility of N. benthamiana to infection following gene silencing was found only for NbGSTU1-silenced plants, which had 130% more lesions and 67% more colonization by C. orbiculare compared with control plants. These results demonstrate that the different GST genes respond in different ways to fungal infection, and at least one plant GST gene has an important role in disease development.

Nosema ceranae is a microsporidian fungus that parasitizes the midgut epithelial cells of honey bees, Apis mellifera. Due to the role that midgut microorganisms play in bee health and immunity, food supplementation with prebiotics and probiotics may assist in the control of N. ceranae. The dietary fiber prebiotics acacia gum, inulin, and fructooligosaccharides, as well as the commercial probiotics Vetafarm Probotic, Protexin Concentrate single-strain (Enterococcus faecium), and Protexin Concentrate multi-strain (Lactobacillus acidophilus, L. plantarum, L. rhamnosus, L. delbrueckii, Bifidobacterium bifidum, Streptococcus salivarius, and E. faecium) were tested for their effect on N. ceranae spore loads and honey bee survivorship. Bees kept in cages were inoculated with N. ceranae spores and single-dose treatments were administered in sugar syrup. Acacia gum caused the greatest reduction in N. ceranae spore numbers (67%) but also significantly increased bee mortality (62.2%). However, Protexin Concentrate single-strain gave similarly reduced spore numbers (59%) without affecting the mortality. In a second experiment, multiple doses of the probiotics revealed significantly reduced spore numbers with 2.50 mg/mL Vetafarm Probotic, and 0.25, 1.25, and 2.50 mg/mL Protexin Concentrate single-strain. Mortality was also significantly reduced with 1.25 mg/mL Protexin Concentrate single-strain. N. ceranae-inoculated bees fed 3.75 mg/mL Vetafarm Probotic had higher survival than N. ceranae-inoculated bees, which was similar to that of non-inoculated bees, while N. ceranae-inoculated bees fed 2.50 mg/mL Protexin Concentrate single-strain, had significantly higher survival than both N. ceranae-inoculated and non-inoculated bees. Protexin Concentrate single-strain is promising as it can reduce N. ceranae proliferation and increase bee survivorship of infected bees, even compared to healthy, non-infected bees.

Two periods of increased ethylene production were observed after inoculation of Nicotiana tabacum by Colletotrichum destructivum. This pathogen exhibits an intracellular hemibiotrophic infection process, with a biotrophic phase followed by a necrotrophic phase. Ethylene production first increased during the biotrophic phase with a peak at 24 h before the necrotrophic phase. A second increase in ethylene occurred late in the necrotrophic phase when the lesions were expanding. Two different 1‐aminocyclopropane‐1‐carboxylic acid synthase genes showed increased expression after the first ethylene peak with a maximum at 24 h before the second ethylene increase. Expression of an 1‐aminocyclopropane‐1‐carboxylic acid oxidase (ACO) gene increased during the first ethylene peak and then declined at the beginning of the second ethylene increase. A second ACO gene showed relatively little change in expression during infection with slightly higher expression at 24 h before the second ethylene increase, and a third ACO gene showed a progressive decline in expression with a major decrease occurring before the second ethylene increase. Inoculation of ethylene‐insensitive tobacco with C. destructivum revealed that it was more susceptible than the wild type. The changes in ethylene production and associated gene expression as well as the increased disease susceptibility of ethylene‐insensitive tobacco indicate that ethylene plays a role in this interaction, perhaps as a signalling molecule to trigger defense mechanisms.

Resistance in Nicotiana benthamiana against anthracnose caused by the hemibiotrophic fungus Colletotrichum orbiculare was activated by benzothiadiazole (BTH), (2R,3R)­butanediol or PC1, an isoparaffin‐based mixture. In inoculation experiments, BTH, (2R,3R)­butanediol and PC1 reduced the number of lesions per leaf area caused by C . orbiculare by 98%, 77% and 81%, respectively. Foliar application of BTH induced expression of genes for the acidic pathogenesis‐related (PR) proteins, NbPR‐1a , NbPR‐ 3 Q and acidic NbPR‐ 5. In contrast, soil application of (2R,3R)­butanediol or PC1 primed expression of genes for the basic PR proteins, NbPRb‐1b , basic NbPR‐2 and NbPR‐5dB . These results are consistent with the activation of salicylic‐acid‐dependent systemic acquired resistance (SAR) by BTH and that of jasmonate/ethylene‐dependent induced systemic resistance (ISR) by (2R,3R)­butanediol or PC1, and show that (2R,3R)­butanediol and PC1 can affect gene expression similarly to plant growth‐promoting rhizobacteria. However, the effects of (2R,3R)‐butanediol and PC1 were not identical. In addition to priming, (2R,3R)‐butanediol induced expression of basic NbPR‐2 , whereas PC1 treatment induced expression of both NbPRb‐1b and basic NbPR‐2 . Although a number of microbial products, such as (2R,3R)­butanediol, have been shown to produce ISR, this is the first demonstration that an isoparaffin‐based mixture, not derived from a microorganism, can produce ISR.

The Ilyonectria radicicola species complex (A.A. Hildebr.) A. Cabral and Crous 2011 contains species of soilborne necrotrophic plant pathogens. The most aggressive to ginseng roots is I. mors-panacis, whereas I. robusta, I. crassa, I. panacis and I. radicicola are less aggressive. Infected ginseng roots show orange-red to black-brown lesions that can expand into a severe root rot, known as disappearing root rot, where only epidermal root tissue remains. Leaves become red-brown with wilting, and stems can have vascular discoloration with black-brown lesions at the base. Less aggressive Ilyonectria species trigger jasmonic acid (JA)-related defenses inducing host ginsenosides, pathogenesis-related (PR) proteins, wound periderm, and cell wall thickening. In contrast, I. mors-panacis triggers reactive oxygen species (ROS) and salicylic acid (SA) production but suppresses JA-related defenses and ginsenoside accumulation. It is also able to suppress SA-related PR protein production. Virulence factors include potential effectors that may suppress PAMP (Pathogen Associated Molecular Patterns) triggered immunity (PTI), polyphenoloxidases, Hsp90 inhibitors, siderophores and cell-wall-degrading enzymes, such as pectinases. Overall, I. mors-panacis appears to be more aggressive because it can suppress JA and SA-related PTI allowing for more extensive colonization of ginseng roots. While many possible mechanisms of host resistance and pathogen virulence mechanisms have been examined, there is a need for using genetic approaches, such as RNAi silencing of genes of Panax or Ilyonectria, to determine their importance in the interaction.