Identification of Phytophthora cinnamomi CRN effectors and their roles in manipulating cell death during Persea americana infection.

The oomycete Phytophthora cinnamomi is a devastating plant pathogen with a notably broad host range. It is the causal agent of Phytophthora root rot (PRR), arguably the most economically important yield-limiting disease in Persea americana (avocado). Despite this, our understanding of the mechanisms P. cinnamomi employs to infect and successfully colonize avocado remains limited, particularly regarding the pathogen's ability to maintain its biotrophic and necrotrophic lifestyles during infection. The pathogen utilises a large repertoire of effector proteins which function in facilitating and establishing disease in susceptible host plants. Crinkling and necrosis effectors (CRN/Crinklers) are suspected to manipulate cell death to aid in maintenance of the pathogens biotrophic and necrotrophic lifestyles during different stages of infection. The current study identified 25 P. cinnamomi CRN effectors from the GKB4 genome using an HMM profile and assigned putative function to them as either cell death inducers or suppressors. Function was assigned to 10 PcinCRNs by analysing their RNA-seq expression profiles, relatedness to other functionally characterised Phytophthora CRNs and tertiary protein predictions. The full-length coding sequences for these PcinCRNs were confirmed by Sanger sequencing, six of which were found to have two divergent alleles. The presence of alleles indicates that the proteins encoded may perform contradicting functions in cell death manipulation, or function in different host plant species. Overall, this study provides a foundation for future research on P. cinnamomi infection and cell death manipulation mechanisms.

Differences in transcriptomic responses upon Phytophthora palmivora infection among cultivars reveal potential underlying resistant mechanisms in durian.

BACKGROUND: Phytophthora palmivora is a devastating oomycete pathogen in durian, one of the most economically important crops in Southeast Asia. The use of fungicides in Phytophthora management may not be a long-term solution because of emerging chemical resistance issues. It is crucial to develop Phytophthora-resistant durian cultivars, and information regarding the underlying resistance mechanisms is valuable for smart breeding programs. RESULTS: In this study, we conducted RNA sequencing (RNA-seq) to investigate early gene expression responses (at 8, 24, and 48 h) after the P. palmivora infection in three durian cultivars, which included one resistant cultivar (Puangmanee; PM) and two susceptible cultivars (Monthong; MT and Kradumthong; KD). We performed co-expression and differential gene expression analyses to capture gene expression patterns and identify the differentially expressed genes. The results showed that genes encoding heat shock proteins (HSPs) were upregulated in all infected durians. The expression levels of genes encoding HSPs, such as ERdj3B, were high only in infected PM. A higher level of P. palmivora resistance in PM appeared to be associated with higher expression levels of various genes encoding defense and chitin response proteins, such as lysM domain receptor-like kinases. MT had a lower resistance level than PM, although it possessed more upregulated genes during P. palmivora infection. Many photosynthetic and defense genes were upregulated in the infected MT, although their expression levels were lower than those in the infected PM. KD, the least resistant cultivar, showed downregulation of genes involved in cell wall organization or biogenesis during P. palmivora infection. CONCLUSIONS: Our results showed that the three durian cultivars exhibited significantly different gene expression patterns in response to P. palmivora infection. The upregulation of genes encoding HSPs was common in all studied durians. The high expression of genes encoding chitin response proteins likely contributed to P. palmivora resistance in durians. Durian susceptibility was associated with low basal expression of defense genes and downregulation of several cell wall-related genes. These findings enhance our understanding of durian resistance to Phytophthora infection and could be useful for the development of elite durian cultivars.

Microbial dynamics and quality characteristics of spontaneously fermented salamis produced by replacing pork fat with avocado pulp.

The aim of this study was to develop a novel and healthier fermented meat product by replacing pork fat with avocado pulp (AVP) during salami production. Experimental salamis were produced under laboratory conditions by substituting pork fat with AVP partially (10-AVP) and totally (20-AVP), while control salamis (CTR) remained AVP-free. The microbial composition of control and experimental salamis was assessed using a combined culture-dependent and -independent approach. Over a 20-days ripening period, lactic acid bacteria, coagulase-negative staphylococci, and yeasts dominated the microbial community, with approximate levels of 9.0, 7.0 and 6.0 log CFU/g, respectively. Illumina technology identified 26 taxonomic groups, with leuconostocs being the predominant group across all trials [constituting 31.26-59.12 % of relative abundance (RA)]. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed changes in fatty acid composition and volatile organic compounds due to the substitution of pork fat with AVP. Specifically, monounsaturated fatty acids and terpene compounds increased, while saturated fatty acids and lipid oxidation products decreased. Although AVP influenced the sensory characteristics of the salamis, the highest overall satisfaction ratings were observed for the 10-AVP salamis. Consequently, substituting pork fat with AVP emerges as a viable strategy for producing healthier salamis and diversifying the meat product portfolio.

Biological Control of Avocado Branch Blight Caused by Lasiodiplodia theobromae Using Bacillus velezensis.

In recent years, avocado branch blight has gradually become one of the major diseases causing mortality of avocado trees, which seriously affects the economic development of avocado planting regions. In order to investigate the cause of the disease, the pathogens were isolated from the interroot of avocado trees with the onset of the disease and identified as Lasiodiplodia theobromae. At the same time, three Bacillus velezensis strains, YK194, YK201, and YK268, with better antagonistic effects and high stability against L. theobromae, were isolated from the rhizospheric soil of healthy avocado plants. The results of branch experiments and field trials showed that the avocado leaves as well as branches treated with the strains YK194, YK201, and YK268 did not develop disease, and the incidence of avocado trees was significantly reduced. In the branch experiments, the biological control effect of the strains YK194, YK201, and YK268 reached 62.07, 52.70, and 72.45%, respectively. In the field experiments, it reached 63.85, 63.43, and 73.86%, respectively, which indicated that all these three strains possessed good biological control effects on avocado branch blight. Further investigation on the mechanism of action of antagonistic strains revealed that B. velezensis YK268 could produce lipopeptides, namely, surfactin, fengycin, and iturin, which could significantly inhibit the spore germination of L. theobromae. Consequently, these three isolates have potential as biocontrol agents against L. theobromae.

Identity, Pathogenicity, and Genetic Diversity of Lasiodiplodia Species Associated with Stem-End Rot of Avocado in China.

Stem-end rot (SER) causes brown necrotic lesions in the pulp near the base of the fruit pedicel and is one of the most devastating postharvest diseases of avocados in all avocado-growing regions of the world. China's avocado industry is growing very rapidly, and the planting area is expanding, but little is known about the pathogens and genetic diversity of avocado SER. To determine the causal agents of SER, avocado fruits were sampled from the main avocado-producing areas in China during 2020 and 2021. Fungal isolates were obtained from SER symptomatic avocado fruits and identified by morphology combined with phylogenetic analysis of internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α), and ß-tubulin (TUB2) gene sequences. All 101 isolates belonged to Lasiodiplodia spp.; four Lasiodiplodia species were identified, namely, L. pseudotheobromae (59.41%), L. theobromae (24.75%), L. mahajangana (7.92%), and L. euphorbiaceicola (1.98%); and six others are classified as Lasiodiplodia sp. (5.94%). There were only slight morphological differences in colonies and conidia of these four species of Lasiodiplodia. The pathogenicity tests showed symptoms of SER, and 92.08% of the isolates exhibited a high level of virulence on avocado (disease index >70), related to the disease severity on avocado fruits. All tested isolates grew well under the temperature ranging from 23 to 33°C. There was a significant difference in mycelial growth between the four species of Lasiodiplodia after treatment with high or low temperatures. The growth of L. pseudotheobromae was the fastest at 13 to 18°C but was the lowest at 38°C (P < 0.05). The red pigment could be produced by all tested isolates after culturing for 7 days at 38°C. The mycelial growth rate was the fastest on PDA medium, and the slowest on the OMA medium but promoted spore formation (P < 0.05). In addition, the genetic diversity of pathogenic Lasiodiplodia species associated with SER collected from avocado, mango, guava, and soursop fruits was determined. A total of 74 isolates were clustered into four main ISSR groups by the unweighted pair-group method with arithmetic mean analysis, and the classification of this group was related to the host. Extensive diversity was detected in the Lasiodiplodia populations. The diverse geographical origins and host species significantly influenced the population differentiation, and most of the genetic variation occurred within populations (P < 0.001). This is the first study to identify the major pathogens of avocado SER in China, survey their occurrence and pathogenicity, and include a comparative analysis of genetic diversity with Lasiodiplodia spp. causing SER on other fruit hosts. Collectively, the Lasiodiplodia species complex affecting avocado showed high pathogenicity and diversity, while L. pseudotheobromae was the most frequently isolated species in China. The results of this study provide insights into the aspects of the epidemic of SER disease caused by Lasiodiplodia species, which will help in developing strategies for the management and control of SER in avocado.

Effects of Exogenous Application of Methyl Jasmonate and Salicylic Acid on the Physiological and Molecular Response of 'Dusa' Avocado to Rosellinia necatrix.

Methyl jasmonate (MeJA) and salicylic acid (SA) are important in mediating plant responses to abiotic and biotic stresses. MeJA and SA can act as elicitors by triggering plant defense responses similar to those induced by pathogens and may even provide long-term protection against them. Thus, exogenous application of MeJA and SA could protect susceptible avocado plants against white root rot (WRR) disease caused by the necrotrophic fungus Rosellinia necatrix, one of the main diseases affecting avocado orchards. This work evaluates the effects of MeJA or SA on the physiological and molecular response of susceptible 'Dusa' avocado rootstock and their ability to provide some protection against WRR. The application of MeJA and SA in avocado increased photoprotective mechanisms (nonphotochemical chlorophyll fluorescence quenching) and upregulated the glutathione S-transferase, suggesting the triggering of mechanisms closely related to oxidative stress relief and reactive oxygen species scavenging. In contrast to SA, MeJA's effects were more pronounced at the morphoanatomical level, including functional traits such as high leaf mass area, high stomatal density, and high root/shoot ratio, closely related to strategies to cope with water scarcity and WRR disease. Moreover, MeJA upregulated a greater number of defense-related genes than SA, including a glu protease inhibitor, a key gene in avocado defense against R. necatrix. The overall effects of MeJA increased 'Dusa' avocado tolerance to R. necatrix by inducing a primed state that delayed WRR disease symptoms. These findings point toward the use of MeJA application as an environmentally friendly strategy to mitigate the impact of this disease on susceptible avocado orchards.

Fungal microbiome shifts on avocado fruit associated with a combination of postharvest chemical and physical interventions.

AIM OF THE STUDY: The aim was to characterize the baseline microbial population of the avocado carposphere and understand shifts in community structure from the harvest to ready-to-eat stages. METHODS AND RESULTS: The changes in surface or stem-end (SE) fungal microbiomes at the postharvest stage of avocado fruit were studied using next-generation sequencing of the internal transcribed spacer region. Avocado fructoplane and SE pulp fungal richness differed significantly between postharvest stages with a decline following prochloraz dip treatments. Known postharvest decay-causing genera, Colletotrichum, Fusarium, Alternaria, Epicoccum, Penicillium and Neofusicoccum were detected, with Papiliotrema, Meyerozyma and Aureobasidium confirmed as the most dominant potentially beneficial genera. Postharvest interventions such as prochloraz had a negative non-target effect on the presence of Papiliotrema flavescens on the avocado fructoplane. CONCLUSION: Our findings reveal a core community of beneficial and pathogenic taxa in the avocado fructoplane and further highlight the reduction of pathogenic fungi as a consequence of fungicide use. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides important baseline data for further exploration of fungal population shifts in avocado fruit driven by chemical (fungicide) as well as physical (cold storage) interventions.

Population Genetic Analyses of Phytophthora cinnamomi Reveals Three Lineages and Movement Between Natural Vegetation and Avocado Orchards in South Africa.

Phytophthora cinnamomi is the causal agent of root rot, canker, and dieback of thousands of plant species around the globe. This oomycete not only causes severe economic losses to forestry and agricultural industries, but also threatens the health of various plants in natural ecosystems. In this study, 380 isolates of P. cinnamomi from four avocado production areas and two regions of natural vegetation in South Africa were investigated using 15 microsatellite markers. These populations were found to have a low level of genetic diversity and consisted of isolates from three lineages. Shared genotypes were detected between isolates from avocado orchards and natural vegetation, indicating the movement of isolates between these areas. The population from the Western Cape natural vegetation had the highest level of genotypic diversity and number of unique alleles, indicating this could be the point of introduction of P. cinnamomi to South Africa. Index of association analysis suggested that five of six populations were under linkage disequilibrium, suggesting a clonal mode of reproduction, whereas genotypes sampled from a recently established avocado orchard in the Western Cape were derived from a randomly recombining population. This study provided novel insights on the genetic diversity and spread of P. cinnamomi in South Africa. It also reported on the predominance of triploidy in natural occurring populations and provided evidence for recombination of P. cinnamomi for the first time. The presence of two dominant genotypes in all avocado production areas in South Africa highlight the importance of considering them in disease management and resistance breeding programs.

The avocado genome informs deep angiosperm phylogeny, highlights introgressive hybridization, and reveals pathogen-influenced gene space adaptation.

The avocado, Persea americana, is a fruit crop of immense importance to Mexican agriculture with an increasing demand worldwide. Avocado lies in the anciently diverged magnoliid clade of angiosperms, which has a controversial phylogenetic position relative to eudicots and monocots. We sequenced the nuclear genomes of the Mexican avocado race, P. americana var. drymifolia, and the most commercially popular hybrid cultivar, Hass, and anchored the latter to chromosomes using a genetic map. Resequencing of Guatemalan and West Indian varieties revealed that ∼39% of the Hass genome represents Guatemalan source regions introgressed into a Mexican race background. Some introgressed blocks are extremely large, consistent with the recent origin of the cultivar. The avocado lineage experienced 2 lineage-specific polyploidy events during its evolutionary history. Although gene-tree/species-tree phylogenomic results are inconclusive, syntenic ortholog distances to other species place avocado as sister to the enormous monocot and eudicot lineages combined. Duplicate genes descending from polyploidy augmented the transcription factor diversity of avocado, while tandem duplicates enhanced the secondary metabolism of the species. Phenylpropanoid biosynthesis, known to be elicited by Colletotrichum (anthracnose) pathogen infection in avocado, is one enriched function among tandems. Furthermore, transcriptome data show that tandem duplicates are significantly up- and down-regulated in response to anthracnose infection, whereas polyploid duplicates are not, supporting the general view that collections of tandem duplicates contribute evolutionarily recent "tuning knobs" in the genome adaptive landscapes of given species.

Evaluation of Fungicide Soil Drench Treatments to Manage Black Root Rot Disease of Avocado.

Four common fungicidal products were evaluated for their effect on symptoms caused by two nectriaceous black root rot fungi, Calonectria ilicicola and Dactylonectria macrodidyma, when applied as pot drenches to avocado (Persea americana) seedlings in the greenhouse. Applications of fludioxonil, thiophanate-methyl + etridiazole, prochloraz, and prochloraz MnCl at 2 and 4 weeks after inoculation with C. ilicicola significantly reduced root necrosis and improved root and aboveground plant biomass compared with water-treated controls. Fludioxonil reduced necrosis by 60% and had a significantly lower frequency of reisolation of C. ilicicola than the other three fungicide treatments. D. macrodidyma inoculation caused less severe symptoms in seedlings than C. ilicicola despite the longer duration of the trial. Pot drenches with fludioxonil, thiophanate-methyl + etridiazole, and prochloraz MnCl, but not prochloraz alone, significantly reduced root necrosis caused by D. macrodidyma. Prochloraz MnCl was the only fungicide treatment to increase root and plant biomass compared with water-treated controls. Both fludioxonil and prochloraz MnCl reduced the frequency of reisolation of D. macrodidyma from necrotic roots by about 50% compared with the other fungicides or water controls. The results indicated that drenches with these fungicides may suppress existing low to moderate black root rot infection, allowing new root growth and improved establishment in the orchard. Fungicide drenching must not replace best-practice disease management strategies in nurseries but may be a useful tool in crisis situations.

Barks from avocado trees of different geographic locations have consistent microbial communities.

Bark is a permanent surface for microbial colonization at the interface of trees and the surrounding air, but little is known about its microbial communities. We used shotgun metagenomic sequencing to analyze the bark microbiomes of avocado trees from two orchards, and compared one of them to rhizospheric soil. It was shown that the microbial communities of avocado bark have a well-defined taxonomic structure, with consistent patterns of abundance of bacteria, fungi, and archaea, even in trees from two different locations. Bark microbial communities were distinct from rhizospheric soil, although they showed overlap in some taxa. Thus, avocado bark is a well-defined environment, providing niches for specific taxonomic groups, many of which are also found in other aerial plant tissues. The present in-depth characterization of bark microbial communities can form a basis for their future manipulation for agronomical purposes.

Non-Chemical Treatments for the Pre- and Post-Harvest Elicitation of Defense Mechanisms in the Fungi-Avocado Pathosystem.

The greatest challenge for the avocado (Persea americana Miller) industry is to maintain the quality of the fruit to meet consumer requirements. Anthracnose is considered the most important disease in this industry, and it is caused by different species of the genus Colletotrichum, although other pathogens can be equally important. The defense mechanisms that fruit naturally uses can be triggered in response to the attack of pathogenic microorganisms and also by the application of exogenous elicitors in the form of GRAS compounds. The elicitors are recognized by receptors called PRRs, which are proteins located on the avocado fruit cell surface that have high affinity and specificity for PAMPs, MAMPs, and DAMPs. The activation of defense-signaling pathways depends on ethylene, salicylic, and jasmonic acids, and it occurs hours or days after PTI activation. These defense mechanisms aim to drive the pathogen to death. The application of essential oils, antagonists, volatile compounds, chitosan and silicon has been documented in vitro and on avocado fruit, showing some of them to have elicitor and fungicidal effects that are reflected in the postharvest quality of the fruit and a lower incidence of diseases. The main focus of these studies has been on anthracnose diseases. This review presents the most relevant advances in the use of natural compounds with antifungal and elicitor effects in plant tissues.

Bacillus mycoides A1 and Bacillus tequilensis A3 inhibit the growth of a member of the phytopathogen Colletotrichum gloeosporioides species complex in avocado.

BACKGROUND: Avocado is affected by Colletotrichum gloeosporioides causing anthracnose. Antagonistic microorganisms against C. gloeosporioides represent an alternative for biological control. Accordingly, in the present study, we focused on the isolation and characterization of potential antagonist bacteria against a member of the C. gloeosporioides species complex with respect to their possible future application. RESULTS: Samples of avocado rhizospheric soil were aquired from an orchard located in Ocuituco, Morelos, Mexico, aiming to obtain bacterial isolates with potential antifungal activity. From the soil samples, 136 bacteria were isolated and they were then challenged against a member of the C. gloeosporioides species complex; only three bacterial isolates A1, A2 and A3 significantly diminished mycelial fungal growth by 75%, 70% and 60%, respectively. Two of these isolates were identified by 16S rRNA as Bacillus mycoides (A1 and A2) and the third was identified as Bacillus tequilensis (A3). Bacillus mycoides bacterial cell-free supernatant reduced the mycelial growth of a member of the C. gloeosporioides species complex isolated from avocado by 65%, whereas Bacillus tequilensis A3 supernatant did so by 25% after 3 days post inoculation. Bacillus tequilensis mycoides A1 was a producer of proteases, indolacetic acid and siderophores. Preventive treatment using a cell-free supernatant of B. mycoides A1 diminished the severity of anthracnose disease (41.9%) on avocado fruit. CONCLUSION: These results reveal the possibility of using B. mycoides A1 as a potential biological control agent. © 2020 Society of Chemical Industry.

[Spatial distribution of anthracnose (Colletotrichum gloeosporioides Penz) in avocado in the State of Mexico, Mexico]. / Distribución espacial de antracnosis (Colletotrichum gloeosporioides Penz) en aguacate en el Estado de México, México.

Persea americana is a species of great nutritional and economic importance for Mexico, however, like any other agricultural crop, it is affected by pests and diseases that limit its worldwide commercialization. The phytopathogenic fungus Colletotrichum gloeosporioides is the causative agent of anthracnose in avocado and manifests itself in the early stages of fruit development as well as in post-harvest and storage, under conditions of high relative humidity (80%) and at temperatures from 20°C, causing losses economic up to 20% of production. Applying geostatistical methods the present study aims to define the spatial distribution of anthracnose in Hass avocado fruits in four municipalities of the State of Mexico during the period from January to June 2017. The results show that the distribution of anthracnose was adjusted to gaussian and exponential models in most, the infestation maps made through the kriging show more than one center of aggregation of the disease, based on it the infested surface was estimated, finding an infestation of more than 50% in the first samples and up to 98% in the samplings belonging to the month of June in all the areas studied.

Transcriptome analysis of the fungal pathogen Rosellinia necatrix during infection of a susceptible avocado rootstock identifies potential mechanisms of pathogenesis.

BACKGROUND: White root rot disease caused by Rosellinia necatrix is one of the most important threats affecting avocado productivity in tropical and subtropical climates. Control of this disease is complex and nowadays, lies in the use of physical and chemical methods, although none have proven to be fully effective. Detailed understanding of the molecular mechanisms underlying white root rot disease has the potential of aiding future developments in disease resistance and management. In this regard, this study used RNA-Seq technology to compare the transcriptomic profiles of R. necatrix during infection of susceptible avocado 'Dusa' roots with that obtained from the fungus cultured in rich medium. RESULTS: The transcriptomes from three biological replicates of R. necatrix colonizing avocado roots (RGA) and R. necatrix growing on potato dextrose agar media (RGPDA) were analyzed using Illumina sequencing. A total of 12,104 transcripts were obtained, among which 1937 were differentially expressed genes (DEG), 137 exclusively expressed in RGA and 160 in RGPDA. During the root infection process, genes involved in the production of fungal toxins, detoxification and transport of toxic compounds, hormone biosynthesis, gene silencing and plant cell wall degradation were overexpressed. Interestingly, 24 out of the 137 contigs expressed only during R. necatrix growth on avocado roots, were predicted as candidate effector proteins (CEP) with a probability above 60%. The PHI (Pathogen Host Interaction) database revealed that three of the R. necatrix CEP showed homology with previously annotated effectors, already proven experimentally via pathogen-host interaction. CONCLUSIONS: The analysis of the full-length transcriptome of R. necatrix during the infection process is suggesting that the success of this fungus to infect roots of diverse crops might be attributed to the production of different compounds which, singly or in combination, interfere with defense or signaling mechanisms shared among distinct plant families. The transcriptome analysis of R. necatrix during the infection process provides useful information and facilitates further research to a more in -depth understanding of the biology and virulence of this emergent pathogen. In turn, this will make possible to evolve novel strategies for white root rot management in avocado.

Mild water stress-induced priming enhance tolerance to Rosellinia necatrix in susceptible avocado rootstocks.

BACKGROUND: White root rot (WRR) disease caused by Rosellinia necatrix is one of the most important threats affecting avocado orchards in temperate regions. The eradication of WRR is a difficult task and environmentally friendly control methods are needed to lessen its impact. Priming plants with a stressor (biotic or abiotic) can be a strategy to enhance plant defense/tolerance against future stress episodes but, despite the known underlying common mechanisms, few studies use abiotic-priming for improving tolerance to forthcoming biotic-stress and vice versa ('cross-factor priming'). To assess whether cross-factor priming can be a potential method for enhancing avocado tolerance to WRR disease, 'Dusa' avocado rootstocks, susceptible to R. necatrix, were subjected to two levels of water stress (mild-WS and severe-WS) and, after drought-recovery, inoculated with R. necatrix. Physiological response and expression of plant defense related genes after drought-priming as well as the disease progression were evaluated. RESULTS: Water-stressed avocado plants showed lower water potential and stomatal limitations of photosynthesis compared to control plants. In addition, NPQ and qN values increased, indicating the activation of energy dissipating mechanisms closely related to the relief of oxidative stress. This response was proportional to the severity of the water stress and was accompanied by the deregulation of pathogen defense-related genes in the roots. After re-watering, leaf photosynthesis and plant water status recovered rapidly in both treatments, but roots of mild-WS primed plants showed a higher number of overexpressed genes related with plant defense than severe-WS primed plants. Disease progression after inoculating primed plants with R. necatrix was significantly delayed in mild-WS primed plants. CONCLUSIONS: These findings demonstrate that mild-WS can induce a primed state in the WRR susceptible avocado rootstock 'Dusa' and reveal that 'cross-factor priming' with water stress (abiotic stressor) is effective for increasing avocado tolerance against R. necatrix (biotic stressor), underpinning that plant responses against biotic and abiotic stress rely on common mechanisms. Potential applications of these results may involve an enhancement of WRR tolerance of current avocado groves and optimization of water use via low frequency deficit irrigation strategies.

Xyleborus volvulus (Coleoptera: Curculionidae): Biology and Fungal Associates.

The ambrosia beetle Xyleborus volvulus Fabricius has been reported as a potential vector of the plant pathogen Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva that is affecting avocado orchards in South Florida. In this study, we examined its life cycle, process of gallery formation, gallery structure, and fungal associates by rearing one generation on avocado sawdust medium under control conditions. The adult foundress excavated a vertical tunnel that constituted the main gallery with a length of 2.5 cm, followed by the construction of up to six secondary galleries with a total length of 4.4 cm. The time period for one generation (egg to adult) was 28 days. Teneral males emerged 3 days after the emergence of the first females. The F1 generation did not significantly contribute to gallery expansion. Four species of Raffaelea and nine yeast species were recovered from galleries and beetles. Raffaelea arxii and Candida berthetii were the most frequent symbionts recovered from new adults and galleries. Candida berthetii dominated during the early stages of the gallery development, whereas R. arxii was most frequent in later stages. Other Raffaelea species were inconsistently isolated from galleries, which suggests a strong association between Xyleborus volvulus and both R. arxii and C. berthetii These results suggest that R. arxii is the primary nutritional symbiont of X. volvulus and that yeast species may be pioneer colonizers that assist with the growth of fungal symbionts.IMPORTANCE Ambrosia beetles cultivate fungi in tunnels bored into weakened host trees. This obligate interaction is required for their survival as beetles feed on these symbiotic fungi, and the fungi benefit from transportation by the beetles. Xyleborus volvulus carries many nonpathogenic symbionts; however, recently the acquisition of Raffaelea lauricola (the causal agent of a lethal vascular disease of lauraceous trees) by this beetle has altered its status from wood degrader to potential pest in avocado. We conducted a study to understand the relationship of this beetle and its fungal associates. Our results show that X. volvulus has a multipartite flexible association with different Raffaelea species. The lack of fidelity in the mutualistic association may explain the acquisition of R. lauricola Knowing the beetle biology and its mutualistic interactions furthers an understanding of the beetle's role as a potential vector and in disease transmission.

Response of the Biocontrol Agent Pseudomonas pseudoalcaligenes AVO110 to Rosellinia necatrix Exudate.

The rhizobacterium Pseudomonas pseudoalcaligenes AVO110, isolated by the enrichment of competitive avocado root tip colonizers, controls avocado white root rot disease caused by Rosellinia necatrix Here, we applied signature-tagged mutagenesis (STM) during the growth and survival of AVO110 in fungal exudate-containing medium with the goal of identifying the molecular mechanisms linked to the interaction of this bacterium with R. necatrix A total of 26 STM mutants outcompeted by the parental strain in fungal exudate, but not in rich medium, were selected and named growth-attenuated mutants (GAMs). Twenty-one genes were identified as being required for this bacterial-fungal interaction, including membrane transporters, transcriptional regulators, and genes related to the metabolism of hydrocarbons, amino acids, fatty acids, and aromatic compounds. The bacterial traits identified here that are involved in the colonization of fungal hyphae include proteins involved in membrane maintenance (a dynamin-like protein and ColS) or cyclic-di-GMP signaling and chemotaxis. In addition, genes encoding a DNA helicase (recB) and a regulator of alginate production (algQ) were identified as being required for efficient colonization of the avocado rhizosphere.IMPORTANCE Diseases associated with fungal root invasion cause a significant loss of fruit tree production worldwide. The bacterium Pseudomonas pseudoalcaligenes AVO110 controls avocado white root rot disease caused by Rosellinia necatrix by using mechanisms involving competition for nutrients and niches. Here, a functional genomics approach was conducted to identify the bacterial traits involved in the interaction with this fungal pathogen. Our results contribute to a better understanding of the multitrophic interactions established among bacterial biocontrol agents, the plant rhizosphere, and the mycelia of soilborne pathogens.

Development of Three Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Calonectria ilicicola, Dactylonectria macrodidyma, and the Dactylonectria Genus in Avocado Roots.

Black root rot of avocado is a severe disease of nursery trees and young orchard transplants, causing tree death within a year after planting. In Australia, key pathogens include species complexes Calonectria ilicicola and Dactylonectria macrodidyma; however, several other Dactylonectria species also cause the disease. Rapid detection of these pathogens in planta is important to speed up implementation of disease management and reduce loss. The purpose of this study was to develop three loop-mediated isothermal amplification (LAMP) diagnostic assays to rapidly identify species within the C. ilicicola and D. macrodidyma complexes and species in the Dactylonectria genus in avocado roots. Primers were designed from ß-tubulin sequence data of C. ilicicola and from histone H3 of D. macrodidyma and the Dactylonectria genus. The LAMP primers were tested for specificity and sensitivity with 82 fungal isolates, which included the target species complexes C. ilicicola and D. macrodidyma; species within the target Dactylonectria genus viz. D. macrodidyma, D. anthuriicola, D. novozelandica, D. pauciseptata, and D. vitis; and isolates of nontarget species, including Calonectria sp., Cylindrocladiella sp., Gliocladiopsis forsbergii, G. peggii, G. whileyi, Ilyonectria sp., Mariannaea sp., Fusarium sp., and Phytophthora cinnamomi. The species-specific LAMP assays were sensitive and specific at DNA concentrations of 1 pg/µl for C. ilicicola and 0.01 ng/µl for D. macrodidyma, whereas the Dactylonectria genus-wide assay was sensitive to 0.1 ng/µl. Detection of C. ilicicola occurred within 10 to 15 or 15 to 30 min when the template was pure DNA or crude extracts obtained from suspending fungal cultures in sterile water, respectively. Detection of D. macrodidyma was between 12 to 29 min with pure DNA and 16 to 30 min with crude extracts. Dactylonectria spp. were detected within 6 to 25 min with pure DNA and 7 to 23 min with crude extracts. The specificity of the assays was found to be dependent on time and isothermal amplification temperature, with optimal specificity occurring in reactions of <30 min and at temperatures of 67°C for C. ilicicola and D. macrodidyma assays and 69°C for Dactylonectria genus-wide assays. The assays were modified to accommodate a DNA extraction step and use of avocado roots as DNA templates. Detection in avocado roots ranged between 12 to 25 min for C. ilicicola, 12 to 26 min for D. macrodidyma, and 14 to 30 min for species in the Dactylonectria genus. The LAMP assays are applicable across multiple agricultural industries, because C. ilicicola, D. macrodidyma, and Dactylonectria spp. are also important pathogens of various crops and ornamental plants.

Detection of White Root Rot in Avocado Trees by Remote Sensing.

White root rot, caused by the soilborne fungus Rosellinia necatrix, is an important constraint to production for a wide range of woody crop plants such as avocado trees. The current methods of detection of white root rot are based on microbial and molecular techniques, and their application at orchard scale is limited. In this study, physiological parameters provided by imaging techniques were analyzed by machine learning methods. Normalized difference vegetation index (NDVI) and normalized canopy temperature (canopy temperature - air temperature) were tested as predictors of disease by several algorithms. Among them, logistic regression analysis (LRA) trained on NDVI data showed the highest sensitivity and lowest rate of false negatives. This algorithm based on NDVI could be a quick and feasible method to detect trees potentially affected by white root rot in avocado orchards.