A new fungal pathogen of the scavenger mite,Tydeus gloveri

A new fungal pathogen, Hirsutella tydeicola, was found causing epizootics in populations of the scavenger mite, Tydeus gloveri, during the summer of 1979 and 1980 on citrus in Florida. The fungus is described in association with its host using light and scanning electron microscopy. H. tydeicola is compared with a closely related species, H. thompsonii, a coexisting pathogen of the citrus rust mite. All attempts to isolate the fungus on various agar media failed.     

Seasonal prevalence of the insect pathogenic fungus Colletotrichum nymphaeae in Brazilian citrus groves under different chemical pesticide regimes

We report an endemic entomopathogenic fungus, known in Brazil as the 'salmão' fungus and identified here as Colletotrichum nymphaeae (Sordariomycetes: Glomerellales), infecting populations of citrus orthezia scale, Praelongorthezia praelonga. The seasonal prevalence of this pathogen in P. praelonga populations was investigated in three commercial citrus groves maintained under different pesticide regimes. Two citrus groves included inundative releases of another insect pathogenic fungus, Lecanicillium longisporum. Natural epizootics were consistently observed, with up to 84% infection rates being recorded during the warm rainy season. Temporal progression of C. nymphaeae-induced disease varied among the three pesticide regimes. Low infection levels from C. nymphaeae were associated with intensive application of broad spectrum pesticides. However, the prevalence of C. nymphaeae followed a density-dependent pattern with insect host abundance, irrespective of the pesticide regime. High proportions of Lecanicillium-infected insects were observed following infection peaks of C. nymphaeae and both fungi together contributed to 95% overall mortality of citrus orthezia during the wet season. Hence, the combined effect of both fungi considerably improves the biological control of citrus orthezia. We also surmise that the host abundance, environmental conditions, and application frequency of chemical pesticides in citrus groves exert a great influence in the seasonal prevalence of C. nymphaeae-induced disease. Altogether, these results suggest that C. nymphaeae is an important pathogen of P. praelonga and indicate that frequent use of synthetic pesticides may delay or reduce fungal epizootics.     

Eggs of Tylenchulus semipenetrans Inhibit Growth of Phytophthora nicotianae and Fusarium solani in vitro

In previous greenhouse and laboratory studies, citrus seedlings infested with the citrus nematode Tylenchulus semipenetrans and later inoculated with the fungus Phylophthora nicotianae grew larger and contained less fungal protein in root tissues than plants infected by only the fungus, demonstrating antagonism of the nematode to the fungus. In this study, we determined whether eggs of the citrus nematode T. semipenetrans and root-knot nematode Meloidogyne arenaria affected mycelial growth of P. nicotianae and Fusarium solani in vitro. Approximately 35,000 live or heat-killed (60°C, 10 minutes) eggs of each nematode species were surface-sterilized with cupric sulfate, mercuric chloride, and streptomycin sulfate and placed in 5-pl drops onto the center of nutrient agar plates. Nutrient agar plugs from actively growing colonies of P. nicotianae or F. solani were placed on top of the eggs for 48 hours after which fungal colony growth was determined. Live citrus nematode eggs suppressed mycelial growth of P. nicotianae and F. solani (P ≤ 0.05) compared to heat-killed eggs and water controls. Reaction of the fungi to heat-killed eggs was variable. Root-knot nematode eggs had no effect on either P. nicotianae or F. solani mycelial growth. The experiment demonstrated a species-specific, direct effect of the eggs of the citrus nematode on P, nicotianae and F. solani.     

Mutations Affecting Hyphal Colonization and Pyoverdine Production in Pseudomonads Antagonistic toward Phytophthora parasitica

In previous studies, Pseudomonas putida 06909 and Pseudomonas fluorescens 09906 suppressed populations of Phytophthora parasitica in the citrus rhizosphere, suggesting that these bacteria may be useful in biological control of citrus root rot. In this study we investigated the mechanisms of antagonism between the bacteria and the fungus. Both bacteria colonized Phytophthora hyphae and inhibited the fungus on agar media. A hyphal column assay was developed to measure the colonization of bacteria on fungal hyphae and to enrich for colonization-deficient mutants. In this way we identified Tn5 mutants of each pseudomonad that were not able to colonize the hyphae and inhibit fungal growth in vitro. Colonization-deficient mutants were nonmotile and lacked flagella. Survival of nonmotile mutants in a citrus soil was similar to survival of a random Tn5 mutant over a 52-day period. Additional screening of random Tn5 mutants of both pseudomonads for loss of fungal inhibition in vitro yielded two distinct types of mutants. Mutants of the first type were deficient in production of pyoverdines and in inhibition of the fungus in vitro, although they still colonized fungal hyphae. Mutants of the second type lacked flagella and were not able to colonize the hyphae or inhibit fungal growth. No role was found for antibiotic production by the two bacteria in the inhibition of the fungus. Our results suggest that both hyphal colonization and pyoverdine production are important in the inhibition of Phytophthora parasitica by P. fluorescens and P. putida in vitro.     

Pilot testing ofKloeckera apiculata for the biological control of postharvest diseases of citrus

The efficacy of the yeastKloeckera apiculata, strain 34-9, in controlling postharvest decay of citrus fruit was evaluated in small-scale and pilot tests in commercial packinghouse. Kloeckera apiculata grew efficiently on different media and maintained its antagonistic activity against spore germination ofPenicillium italicum. In small-scale experiments with citrus fruits dipped in the yeast cell suspension, the development of decay in citrus was effectively inhibited. The yeast was compatible with a mixture of low concentration of a commonly chemical fungicide. In packinghouse tests, combining the yeast with 40 mg/kg Carbendazim (MBC) resulted a reduction in the incidence of decay to a level equal to that of the commercial treatment of 200 mg/kg MBC. The efficacy of the strain 34-9 could also be maintained under packinghouse conditions at a cell concentration of the yeast antagonist as low as 10⁶ cells/ml. No significant difference in the efficacy ofK. apiculata was found in either the drench or the spray application systems tested in citrus packinghouse. Scanning electron microscopy revealed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of strain 34-9 against citrus blue mould may be related to its capability to compete withPenicillium italicum, for space and nutrients and /or involvement of directly antagonist of the yeast on the fungus.     

6,7-Dimethoxycoumarin,a citrus phytoalexin conferring resistance against Phytophthora gummosis

6,7-Dimethoxycoumarin was isolated from the bark of citrus trunks, branches and fruit peels following inoculation with the fungus Phytophthora citrophthora. The compound inhibited growth in vitro of Phytophthora citrophthora, Verticillium dahliae, Penicillium digitatum, P. italicum, Colletotrichum gloeosporioides, Diplodia natalensis and Hendersonula toruloidea.     

Observations on the entomopathogenic fungus Hirsutella citriformis attacking adult Diaphorina citri (Hemiptera: Psyllidae) in a managed citrus grove

A two-year field study was conducted in an orange grove in the United States (Florida) to characterize the phenology of the entomopathogen Hirsutella citriformis Speare infecting adults of the Asian citrus psyllid, Diaphorina citri Kuwayama. On the average, 23% of adults observed on mature leaves were killed by H. citriformis. These dead psyllids were characterized as being mummified and covered to various extents by synnemata produced by the fungus. Mummified cadavers were most abundant on citrus leaves during the fall and winter months, with the maximal percentage of mummified psyllids sometimes exceeding 75% of the total number of adults observed. Mummified cadavers were nearly absent each spring, presumably because relative humidity levels were suboptimal for the fungus at this time. Based on dispersion analyses, a monitoring plan for mummified cadavers would best include multiple samples in individual trees as well as multiple tree samples throughout a grove. Mummified cadavers with synnemata, which serve as point sources for new infections of the fungus in psyllids, were observed to remain on leaves for a mean of 68?days (one cadaver remained on a leaf for 168?days). Rainfall was positively correlated with the number of days mummies remained on leaves while mean daily air temperature was negatively correlated. Mummified cadavers were abundant in the summer during 2006 but not during 2007. This may have been a density-dependent consequence of low psyllid host populations in the grove in 2007. Alternatively, combination sprays of oil and copper applied during 2007 may have suppressed the fungus. This latter possibility prompted a laboratory investigation into the toxicity to H. citriformis of six chemicals commonly used in citrus. Copper hydroxide, petroleum oil, and elemental sulfur at maximum label rates each significantly reduced the infectivity of a laboratory culture of H. citriformis while copper sulfate pentahydrate, aluminum tris and alpha-keto/humic acids did not. This finding indicates that citrus growers interested in capitalizing on H. citriformis as a biological control agent of D. citri should avoid applying high rates of copper hydroxide, oil or sulfur.     

Evaluation of Aspergillus aculeatus GC-09 for the biological control of citrus blue mold caused by Penicillium italicum

Blue mold caused by Penicillium italicum is a severe postharvest disease in citrus fruits. In this study, the fermentation product (FP-E) of Aspergillus aculeatus GC-09, an endophytic fungus isolated from a citrus plant, was found to exhibit antifungal activity against P. italicum with a MIC of 0.3125 mg/mL. The fungus A. aculeatus GC-09 was identified based on the studies of morphology and ITS nucleotide sequence. FP-E significantly inhibited the spore germination and mycelial growth of P. italicum. Scanning electron microscopy (SEM) results of P. italicum treated with FP-E showed shrunken, distorted and collapsed hyphae and conidiospores, indicative of the cell membrane damage, which was further confirmed by the propidium iodide (PI) fluorescent staining analysis. Consistent with the microscopy observation, FP-E led to the leakage of cellular constituents from P. italicum, which is evident from the increase in electrical conductivity and nucleic acid contents in the mycelial solution incubated with FP-E. In addition, FP-E treatment considerably increased the intracellular reactive oxygen species (ROS) content, and reduced the enzyme activities of both catalase (CAT) and peroxidase (POD) in P. italicum cells. Furthermore, orange fruits treated with FP-E showed fewer disease symptoms compared to the untreated fruits. These results suggested that the antifungal activity of FP-E might be associated with the disruption of cell membrane integrity, the accumulation of ROS level, and the reduction of the antioxidant enzymes activity of P. italicum. Therefore, A. aculeatus GC-09 might be a potential microbial resource for the biocontrol of citrus postharvest blue mold.     

Host-Specific Effects of Toxin from the Rough Lemon Pathotype of Alternaria alternata on Mitochondria

Host-specific toxin from the rough lemon (Citrus jambhiri Lush) pathotype of Alternaria alternata (ACR toxin) was tested for effects on mitochondria isolated from several citrus species. The toxin caused uncoupling of oxidative phosphorylation and changes in membrane potential in mitochondria from leaves of the susceptible host (rough lemon); the effects differed from those of carbonylcyanide-m-chlorophenylhydrazone, a typical protonophore. ACR toxin also inhibited malate oxidation, apparently because of lack of NAD⁺ in the matrix. In contrast, the toxin had no effect on mitochondria from citrus species (Dancy tangerine and Emperor mandarin [Citrus reticulata Blanco], and grapefruit [Citrus paradisi Macf.]) that are not hosts of the fungus. The effects of the toxin on mitochondria from rough lemon are similar to the effects of a host-specific toxin from Helminthosporium maydis (HMT) on mitochondria from T-cytoplasm maize. Both ACR and HMT toxins are highly selective for the respective host plants. HMT toxin and methomyl had no effect (toxic or protective) on the activity of ACR toxin against mitochondria from rough lemon.     

Protective effect of Bacillus amyloliquefaciens against infections of Citrus aurantium seedlings by Phoma tracheiphila

Isolate TEB1 an antagonistic endophytic bacterium, obtained from citrus leaves and identified as Bacillus amyloliquefaciens by 16S rDNA sequencing, was used for the biological control of mal secco disease of Citrus aurantium seedlings caused by the mitosporic fungus Phoma tracheiphila. The isolate TEB1 exhibited a good in vitro activity against P. tracheiphila in dual cultures as well as with the well diffusion method. C. aurantium seedlings watered with a suspension of TEB1 cells showed a reduction of 53.61 and 48.63 % in disease severity and incidence, respectively. A PCR test with specific primers was performed 365 days after inoculation and P. tracheiphila was detected along the whole stem in inoculated control plant while no amplification product was obtained in TEB1 treated seedlings. Molecular analysis of TEB1 revealed a positive amplification of fenD and ituC genes responsible of the biosynthesis of fengycin and iturin lipopeptides, respectively. Moreover, observations by optical microscope showed that TEB1 reduced by 55 % the germination of P. tracheiphila conidia and exhibited a marked effect on mycelia structure. Data suggest that lipopeptides produced by the bacterium interact with the cytoplasmic membrane of the fungus causing pore formation. TEB1 appears a potential candidate for the biological control of citrus mal secco disease.     

Lack ofPer os toxicity or pathogenicity in rats fed the fungusHirsutella thompsonii

Symptoms of toxicity or pathogenicity were not observed in white rats fed either whole-culture broth or mycelia of the parasitic fungusHirsutella thompsonii. This fungus is presently under consideration as a microbial control agent of the citrus rust mite,Phyllocoptruta oleivora.     

Penicillium digitatum infection mechanisms in citrus: What do we know so far?

Penicillium digitatum is the major source of postharvest decay in citrus fruits worldwide. This fungus shows a limited host range, being able to infect mainly mature fruit belonging to the Rutaceae family. This highly specific host interaction has attracted the interest of the scientific community. Researchers have investigated the chemical interactions and specialized virulence strategies that facilitate this fungus's fruit colonization, thereby leading to a successful citrus infection. There are several factors that mediate and affect the interaction between P. digitatum and its host citrus, including hydrogen peroxide modulation, secretion of organic acids and consequently pH control, and other strategies described here. The recently achieved sequencing of the complete P. digitatum genome opened up new possibilities for exploration of the virulence factors related to the host-pathogen interaction. Through such techniques as RNAseq, RT-PCR and targeted gene knockout mediated by Agrobacterium tumefaciens, important genes involved in the fungal infection process in citrus have been reported, helping to elucidate the molecular mechanisms, metabolites and genetic components that are involved in the pathogenicity of P. digitatum. Understanding the infection process and fungal strategies represents an important step in developing ways to protect citrus from P. digitatum infection, possibly leading to more productive citriculture.     

Gibberellin-like Substances from the Developing Banana Fruit

The occurrence of 2 gibberellin-like substances was demonstrated in the developing banana fruit, Musa sapientum, Linn. Chemical and biological evidence led to the tentative identification of the 2 compounds as GA₇ and GA_x (previously isolated from citrus fruits). Support for such identification was obtained from thin layer chromatography, gradient elution column chromatography, spectrofluorometry, the dwarf maize test, and the cucumber hypocotyl test. Significance of the GA_x -designated compound increased since it is believed to occur in the fungus Fusarium moniliforme, Sheld. in addition to 2 different species of higher plants. It does not resemble any of the known gibberellins as far as chromatography is concerned.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.     

Isolation and characterization of an Isaria fumosorosea isolate infecting the Asian citrus psyllid in Florida

A fungal pathogen that killed adult Diaphorina citri Kuwayama (Asian citrus psyllid) (Hemiptera: Psyllidae) in Florida citrus groves during the fall of 2005 was identified and characterized. Investigation of this pathogen is important because D. citri vectors citrus greening disease (Huanglongbing), which was reported in Florida in 2005. The morphological and genetic data generated herein support identification of the fungus as Isaria fumosorosea Wize (Ifr) (=Paecilomyces fumosoroseus) (Hypocreales: Cordycipitaceae) from the Asian citrus psyllid (Ifr AsCP). Koch’s postulates were fulfilled after the fungus was isolated in vitro and transmitted to healthy psyllids, which then exhibited a diseased-phenotype similar to that observed in the field. Both in vitro growth characteristics and two Ifr AsCP-specific molecular markers discriminated the psyllid pathogen from another local Ifr isolate, Ifr 97 Apopka. These molecular markers will be useful to track the dynamics of this disease in D. citri populations. The potential for utilizing Ifr to complement existing psyllid pest management strategies is discussed.     

Infection of Citrus Roots by Tylenchulus semipenetrans Reduces Root Infection by Phytophthora nicotianae

Bioassays and whole-plant experiments were conducted to investigate the interaction between Tylenchulus semipenetrans and Phytophthora nicotianae. Both organisms are parasites of the citrus fibrous root cortex. Nematode-infected and non-infected root segments were excised from naturally infected field roots and placed on water agar in close proximity to agar plugs of P. nicotianae and then transferred to a Phytophthora-selective medium. At 10 and 12 days, 50% fewer nematode-infected segments were infected by P. nicotianae than non-infected segments. In whole-plant experiments in glass test tubes, sour orange seedlings were inoculated with two densities (8,000 or 80,000 eggs and second-stage juveniles) of T. semipenetrans, and after establishment of infection were inoculated with two densities (9,000 and 90,000 zoospores) of P. nicotianae. In the first experiment, fungal protein was 53% to 65% lower in the roots infected by both organisms than in roots infected by the fungus only. Compared to plants infected only by P. nicotianae, shoot weights were 33% to 50% greater (P ≤ 0.05) in plants infected by both parasites, regardless of inoculum density. Fibrous and tap root weights were 5% to 23% and 19% to 34% greater (P ≤ 0.05), respectively, in nematode-fungus combination treatments compared to the fungus alone. A second experiment was conducted, where plants were infected by the fungus, the nematode, both organisms, or neither organism. The soil mixture pH for 50% of the plants was adjusted from 4.5 to 7.0 to favor nematode infection. A higher rate of nematode infection of plants growing at pH 7.0 compared to pH 4.5 resulted in greater suppression of fungal development and greater inhibition of fungal damage to the plant. Compared to plants infected only by P. nicotianae, shoot and root weights were 37% and 33% greater (P ≤ 0.05), respectively, in plants infected by both parasites. These experiments have revealed antagonism between T. semipenetrans and P. nicotianae in citrus.     

Development of Microsatellite Markers and Analysis of Genetic Diversity and Population Structure of Colletotrichum gloeosporioides from Ethiopia

Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus, Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (F_ST = 0.049) as evidenced by high level of gene flow estimate (N_m = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.     

The role as inoculum sources of Xanthomonas citri pv. citri surviving on the infected Satsuma mandarin fruits

Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solution II). It indicated that the pathogenicity of the citrus canker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solution directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result spports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.     

Gluconacetobacter sp. gel_SEA623-2, bacterial cellulose producing bacterium isolated from citrus fruit juice

Cellulose producing bacterial strain was isolated from citrus fruit juice fungus. The isolated strain was identified as Gluconacetobacter sp. gel_SEA623-2 based on several morphological characteristics, biochemical tests, and 16S rRNA conducted. Culture conditions for bacterial cellulose production by SEA623-2 were screened in static trays. Conditions were extensively optimized by varying the kind of fruit juice, pH, sugar concentration, and temperature for maximum cellulose production. SEA623-2 has a high productive capacity in citrus processing medium, but not in other fruits. The optimal combination of the media constituents for bacterial cellulose production is as follows: 10% citrus juice, 10% sucrose, 1% acetic acid, and 1% ethanol at 30 °C, pH 3.5. Bacterial cellulose produced by SEA623-2 has soft physical properties, high tensile strength, and high water retention value. The cellulose produced by the selected bacteria is suitable as a cosmetic and medical material.