Encapsulation of wild oregano, Plectranthus amboinicus (Lour.) Spreng, phenolic extract in baker's yeast for the postharvest control of anthracnose in papaya.

BACKGROUND: Anthracnose caused by Colletotrichum gloeosporioides is considered as a major postharvest disease affecting many fruits. This plant disease is traditionally managed with synthetic fungicides, which are generally toxic and are linked to pathogen resistance. Recently, microencapsulated bioactives have been developed as potential alternative strategies to these methods, while utilizing natural fungicides and other phytochemicals. Wild oregano, Plectranthus amboinicus (Lour.) Spreng, contains potent antimicrobial phenolics, but these compounds are volatile and relatively unstable, which limits their efficacy during application. Herein, a baker's yeast microencapsulation system was applied to improve the stability of wild oregano phenolic extract (WOPE) and enhance its antifungal activity against anthracnose. RESULTS: Encapsulation of WOPE in plasmolyzed yeast cells afforded a high encapsulation efficiency (93%) and yielded WOPE-loaded yeast microcapsules (WLYMs) with an average diameter of 2.65 µm. Storage stability studies showed WLYMs are stable for at least 4 months. A 24 -h in vitro release experiment showed that WLYMs had an initial burst release upon redispersion in water, followed by a controlled release to about 80% of the loaded WOPE. Upon application as a spray-type postharvest treatment for papaya, WLYMs exhibited a significantly improved mycelial inhibitory action against C. gloeosporioides and greatly reduced the anthracnose symptoms in papaya fruits. CONCLUSION: This study presented a yeast microencapsulation system that can effectively stabilize WOPE and enhance its antifungal activity, making this microparticle formulation a promising environmentally safe postharvest treatment option to combat anthracnose symptoms in papaya fruits. © 2022 Society of Chemical Industry.

Comparative genomics identifies the Magnaporthe oryzae avirulence effector AvrPi9 that triggers Pi9-mediated blast resistance in rice.

We identified the Magnaporthe oryzae avirulence effector AvrPi9 cognate to rice blast resistance gene Pi9 by comparative genomics of requisite strains derived from a sequential planting method. AvrPi9 encodes a small secreted protein that appears to localize in the biotrophic interfacial complex and is translocated to the host cell during rice infection. AvrPi9 forms a tandem gene array with its paralogue proximal to centromeric region of chromosome 7. AvrPi9 is expressed highly at early stages during initiation of blast disease. Virulent isolate strains contain Mg-SINE within the AvrPi9 coding sequence. Loss of AvrPi9 did not lead to any discernible defects during growth or pathogenesis in M. oryzae. This study reiterates the role of diverse transposable elements as off-switch agents in acquisition of gain-of-virulence in the rice blast fungus. The prevalence of AvrPi9 correlates well with the avirulence pathotype in diverse blast isolates from the Philippines and China, thus supporting the broad-spectrum resistance conferred by Pi9 in different rice growing areas. Our results revealed that Pi9 and Piz-t at the Pi2/9 locus activate race specific resistance by recognizing sequence-unrelated AvrPi9 and AvrPiz-t genes, respectively.

Identification of a hidden resistance gene in tetraploid wheat using laboratory strains of Pyricularia oryzae produced by backcrossing.

In the process (BC3F1 generation) of backcrossing an Avena isolate of Pyricularia oryzae with a Triticum isolate, color mutants with white mycelia were obtained. These white mutants lacked virulence on all (31/31) hexaploid and most (28/32) tetraploid wheat lines tested. In a BC4F1 population, white and black cultures segregated in a 1:1 ratio, suggesting that the mutant phenotype is controlled by a single gene. Furthermore, the mycelial color was perfectly linked with avirulence in the BC4F1 population; white cultures were all avirulent on common wheat (Triticum aestivum) 'Norin 4' (N4) whereas black cultures were all virulent. White cultures in the BC3F1 and BC4F1 generations were also avirulent on tetraploid wheat (T. dicoccoides) accession 'KU109' (Tat4), which was susceptible to all cultures derived from the parental wild isolates through the BC2F1 generation. A cross between Tat4 and a susceptible tetraploid (T. paleocolchicum) accession 'KU196' (Tat14) produced resistant and susceptible F2 seedlings in a 3:1 ratio against the white cultures. In the F3 generation homozygous resistant/segregating/homozygous susceptible lines segregated in a 1:2:1 ratio. These results suggest that the resistance of Tat4 to the white cultures is controlled by a single major gene. This gene, tentatively designated as RmgTd(t), is considered to be a hidden resistance gene because it was not detected with the Br58, F1, BC1F1, or BC2F1 cultures. Cytological analysis revealed that the moderate resistance controlled by RmgTd(t) was associated with a hypersensitive reaction of mesophyll cells.

Phylogenetic analysis, fumonisin production and pathogenicity of Fusarium fujikuroi strains isolated from rice in the Philippines.

BACKGROUND: Fusarium fujikuroi Nirenberg is a maize and rice pathogen causing important agricultural losses and produces fumonisins - mycotoxins which pose health risk to humans and farm animals. However, little information is available about the phylogenetics of this species and its ability to produce fumonisins in rice. We studied 32 strains isolated from rice in the Philippines and performed a phylogenetic analysis using the partial sequence of Elongation Factor 1 alpha (EF-1α) including isolates belonging to closely related species. Fumonisin B1 (FB1 ) production was analyzed in 7-day-old cultures grown in fumonisin-inducing medium by an enzyme-linked immunosorbent assay-based method and by real-time reverse transcriptase-polymerase chain reaction using primers for FUM1 gene, a key gene in fumonisin biosynthesis. RESULTS: Nucleotide diversities per site (π) were 0.00024 ± 0.00022 (standard deviation) for the 32 F. fujikuroi strains from the Philippines and 0.00189 ± 0.00143 for all 34 F. fujikuroi strains, respectively. F. fujikuroi isolates grouped into one cluster separated from the rest of isolates belonging to the closely related F. proliferatum and showed very low variability, irrespective of their geographic origin. The cluster containing strains of F. proliferatum showed higher intraspecific variability than F. fujikuroi. Thirteen of the 32 strains analyzed were FB1 producers (40.62%), with production ranging from 0.386 to 223.83 ppm. All isolates analyzed showed FUM1 gene expression above 1 and higher than the CT value of the non-template control sample. Both seedling stunting and elongation were induced by the isolates in comparison with the control. CONCLUSION: F. fujikuroi are distinct from F. proliferatum isolates based on phytogenetic analysis and are potential fumonisin producers because all are positive for FUM1 gene expression. No relationship between fumonisin production and pathogenicity could be observed.

Screening for Resistance in Selected Tomato Varieties against the Root-Knot Nematode Meloidogyne incognita in the Philippines Using a Molecular Marker and Biochemical Analysis.

Tomato is a high-value vegetable crop widely cultivated in the Philippines, and its production is threatened by various stresses including infection by the root-knot nematode M. incognita. In this study, we checked for resistance to M. incognita in selected tomato germplasm collections and commercially available varieties using a bioassay method, the molecular marker Mi23 and biochemical analysis. Among the eight varieties tested, none showed a resistant reaction against M. incognita. Use of the molecular marker Mi23 yielded 430 bp in all the tomato varieties screened. Phylogenetic analysis using the neighbor-joining method revealed the clustering of consensus sequence of the varieties tested with the susceptible variety S. lycopersicum cv. M82-1-8 and a wild relative, S. pimpinellifolium isolate LA2184. The biochemical analysis showed varying responses among the varieties when they were inoculated with M. incognita. Increased levels of total antioxidant activity were observed in Diamante Max F1, Ilocos Red and Tm 2016 11-1, while total phenolic content was found to be elevated in Athena, Avatar TY F1 and Rosanna. Increased levels of ascorbic acid were observed in Athena and Avatar TY F1 even at 45 dpi. Even though these varieties showed elevated levels of the abovementioned biochemical parameters related to a resistance reaction, all of them showed highly susceptible reactions. Hence, this study showed that these tomato varieties have no resistance against M. incognita and that there is a need to identify other sources of resistance against M. incongita and produce resistant tomato cultivars adapted to local conditions.

Weed-Associated Fungal Endophytes as Biocontrol Agents of Fusarium oxysporum f. sp. cubense TR4 in Cavendish Banana.

The antagonistic activity of fungal endophytes isolated from weeds growing in Cavendish banana farms was determined against Fusarium oxysporum f. sp. cubense TR4 (Foc TR4) causing Fusarium wilt of Cavendish banana. Forty-nine out of the total 357 fungal endophytes from the roots of weeds exhibited antagonistic activity against Foc TR4. High inhibitory activity at 79.61-99.31% based on dual culture assay was recorded in endophytes Lasiodiplodia theobromae TDC029, Trichoderma asperellum TDC075, Ceratobasidium sp. TDC037, Ceratobasidium sp. TDC241, and Ceratobasidium sp. TDC474. All five endophytes were identified through DNA sequencing with 86-100% identity. Endophyte-treated Grand Naine and GCTCV 218 plantlets showed significantly lower disease incidence (p = 0.014), significantly lower degree of leaf yellowing (p = 0.037) and rhizome discoloration (p = 0.003). In addition, the cultivar Grand Naine was consistently highly susceptible compared with the tolerant cultivar GCTCV 218.

Mycotoxigenic Fungi and Mycotoxins in Agricultural Crop Commodities in the Philippines: A Review.

The tropical, warm, and humid conditions that are favorable to the growth and development of mycotoxigenic fungi put the Philippines at a high risk of mycotoxin contamination. To date, seven mycotoxigenic Aspergillus species, four Fusarium species, and one Penicillium species have been isolated from various agricultural crop commodities in the country. There are five mycotoxin groups (aflatoxin, fumonisin, ochratoxin, nivalenol, and zearalenone) that have been detected in both the raw form and the by-products of major crops grown in the country. Since the first scientific report of aflatoxin contamination in the Philippines in 1972, new information has been generated on mycotoxins and mycotoxigenic fungi, but little has been known of other mycotoxins until the last two decades. Further, despite the increase in the understanding of mycotoxigenic fungi and mycotoxins in the country, very limited knowledge exists on practices and measures that control both the fungi and the toxins. This paper reviews the current literature on mycotoxigenic fungi and mycotoxins in the Philippines with emphasis on the last two decades and on other mycotoxins.

Phenotypic and Molecular Analyses of Rhizoctonia spp. Associated with Rice and Other Hosts.

Forty-two Rhizoctonia isolates were collected from rice, mung bean, and grasses from Laguna, Philippines. Sixteen isolates were binucleate Rhizoctonia (BNR), while 26 were multinucleate Rhizoctonia (MNR). BNR isolates produced white to brown, small sclerotia (<1.0 mm) except for mung bean isolates. Twenty MNR isolates produced big (>1.0 mm), light to dark brown sclerotia, three produced salmon-colored masses in the medium, and three did not produce sclerotia. Twenty-three MNR isolates were identified as R. solani AG1-IA using specific primers. Deduced Internal Transcribed Spacer (ITS) sequences of BNR isolates D1FL, NVL, and ScNL shared 100, 97, and 100% identity with R. oryzae-sativae, respectively, while MNR isolates BMgL, IbMgL, and MaSL that produced salmon-colored masses shared 100, 90, and 100% identity with R. oryzae, respectively. Preliminary analysis of the DNA fingerprint patterns generated by repetitive-element PCR (rep-PCR) clustered the 42 isolates into three: R. solani, R. oryzae-sativae, and R. oryzae, together with Ceratobasidium sp. R. solani isolates were pathogenic on rice (TN1), barnyard grass, mungbean (Pagasa 3), and tomato (Athena), while R. oryzae and R. oryzae-sativae isolates were only pathogenic on rice, Echinochloa crus-galli, and tomato. R. solani and R. oryzae were found to be more virulent than R. oryzae-sativae.

Non-Synergistic Effect of Trichoderma harzianum and Glomus spp. in Reducing Infection of Fusarium Wilt in Banana.

Philippine banana is currently threatened by Fusarium oxysporum f. sp. cubense Tropical Race 4 (FocR4). This study investigated the use of Trichoderma harzianum pre-treated with Glomus spp, as a means of managing Fusarium wilt on young 'Lakatan' banana seedlings. Results showed that Glomus applied basally significantly improved banana seedling growth with increased increment in plant height and pseudostem diameter and heavier root weight. The application of Glomus spp. alone offered 100% protection to the 'Lakatan' seedlings against FocR4 as indicated by the absence of the wilting symptom. A combination of T. harzianum and Glomus spp. also gave significant effect against Fusarium wilt through delayed disease progression in the seedlings but was not synergistic. Competitive effects were suspected when application of the two biological control agents on banana roots was done simultaneously.

Genetic Diversity of Fusarium oxysporum f. sp. cubense Causing Panama Wilt of Banana in the Philippines.

Panama wilt, caused by Fusarium oxysporum f. sp. cubense (Foc) is considered one of the most devastating banana diseases in recorded history. The disease threatens the banana industry due to Tropical Race 4 (TR4) infecting the Cavendish cultivar. Forty-two of the 45 representative isolates from Luzon were pathogenic, based on leaf symptom index and vascular discoloration rating. Accurate, fast and reliable identification are pre-requisites for effective management considering there are yet no proven effective chemicals to control the disease, thus the confirmation by a PCR-based diagnostic tool is essential. Using race-specific primers, FocTr4-F/FocTr4-R and Foc-1/Foc-2, the absence of TR4 in Luzon has been confirmed, however, the occurrence of Race 4 has been reported, which should also be taken in consideration as the latter can also cause severe damage under favorable conditions. Furthermore, to examine genetic diversity of Foc in bananas, 55 of the 164 isolates collected from Regions I, II, III, IV and Cordillera Administrative Region (CAR) were analyzed by fingerprinting techniques using M13, ERIC and REP primers. Twenty-two reference isolates from Mindanao were also analyzed using the same primers. Foc isolates were differentiated into two clades at 25% similarity level, classifying all Mindanao isolates to clade A. Consistently high genetic variation was obtained from Luzon isolates using M13, an arbitrarily primed fingerprinting technique and repetitive elements, REP and ERIC-PCR, while low genetic variation was obtained from Mindanao isolates. ERIC-PCR was the most informative and predictive fingerprinting method as the TR4 isolates from Mindanao were grouped together. No grouping of Foc isolates was observed with respect to geographical origin, except isolates from Mindanao. In addition, grouping of Foc4 is also regardless of host variety in all analyses conducted. Overall, high genetic variability was recorded in Foc Philippine population for the three primers used, which might render host resistance vulnerable.

Geographic Distribution of Avirulence Genes of the Rice Blast Fungus Magnaporthe oryzae in the Philippines.

A total of 131 contemporary and 33 reference isolates representing a number of multi-locus genotypes of Magnaporthe oryzae were subjected to a PCR test to detect the presence/absence of avirulence (Avr) genes. Results revealed that the more frequently occurring genes were Avr-Pik (81.50%), Avr-Pita (64.16%) and Avr-Pii (47.98%), whereas the less frequently occurring genes were Avr-Pizt (19.08%) and Avr-Pia (5.20%). It was also laid out that the presence of Avr genes in M. oryzae is strongly associated with agroecosystems where the complementary resistant (R) genes exist. No significant association, however, was noted on the functional Avr genes and the major geographic locations. Furthermore, it was identified that the upland varieties locally known as "Milagrosa" and "Waray" contained all the R genes complementary to the Avr genes tested.

Female fertility and mating type distribution in a Philippine population of Fusarium verticillioides.

Fusarium verticillioides is a common causal agent of maize ear rot in the Philippines. Eighty isolates were collected from healthy and infected maize cobs from Laguna province. Fifty isolates crossed with one of the mating type A testers. The ratio of MATA-1:MATA-2 is 27:23, which follows a Mendelian ratio of 1:1. The effective population number, Ne, was determined by mating type and male/hermaphrodite polymorphisms. The effective population number for mating type, Ne mt, is 81% of the count (total population), and that for male/hermaphrodite status, Ne f, is 36-42% of the count (total population). Sexual reproduction in Philippine isolates of F. verticillioides does not occur frequently, compared to F. verticillioides in other regions of the world.

Assessment of mycotoxin risk on corn in the Philippines under current and future climate change conditions.

This study attempts to assess the risk of mycotoxins (aflatoxins and fumonisins) contamination on corn in the Philippines under current and projected climate change conditions using fuzzy logic methodology based on the published range of temperature and rainfall conditions that favor mycotoxin development. Based on the analysis, projected climatic change will reduce the risk of aflatoxin contamination in the country due to increased rainfall. In the case of fumonisin contamination, most parts of the country are at a very high risk both under current conditions and the projected climate change conditions.

Genetic Mapping of Pathogenicity and Aggressiveness of Gibberella zeae (Fusarium graminearum) Toward Wheat.

ABSTRACT Gibberella zeae is the major fungal pathogen of Fusarium head blight of wheat and produces several mycotoxins that are harmful to humans and domesticated animals. We identified loci associated with pathogenicity and aggressiveness on an amplified fragment length polymorphism based genetic map of G. zeae in a cross between a lineage 6 nivalenol producer from Japan and a lineage 7 deoxynivalenol producer from Kansas. Ninety-nine progeny and the parents were tested in the greenhouse for 2 years. Progeny segregated qualitatively (61:38) for pathogenicity:nonpathogenicity, respectively. The trait maps to linkage group IV, which is adjacent to loci that affect colony pigmentation, perithecium production, and trichothecene toxin amount. Among the 61 pathogenic progeny, the amount of disease induced (aggressiveness) varied quantitatively. Two reproducible quantitative trait loci (QTL) for aggressiveness were detected on linkage group I using simple interval analysis. A QTL linked to the TRI5 locus (trichodiene synthase in the trichothecene pathway gene cluster) explained 51% of the variation observed, and a second QTL that was 50 centimorgans away explained 29% of the phenotypic variation. TRI5 is tightly linked to the locus controlling trichothecene toxin type. The two QTLs, however, were likely part of the same QTL using composite interval analysis. Progeny that produced deoxynivalenol were, on average, approximately twice as aggressive as those that produced nivalenol. No transgressive segregation for aggressiveness was detected. The rather simple inheritance of both traits in this interlineage cross suggests that relatively few loci for pathogenicity or aggressiveness differ between lineage 6 and 7.

Molecular Quantification and Genetic Diversity of Toxigenic Fusarium Species in Northern Europe as Compared to Those in Southern Europe.

Fusarium species produce important mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV) and T-2/HT-2-toxins in cereals. The highest DON and T-2/HT-2 toxin levels in northern Europe have been found in oats. About 12%-24% of Finnish oat samples in 2012 contained >1.75 mg·kg-1 of DON, which belongs to type B trichothecenes. Fusarium graminearum is the most important DON producer in northern Europe and Asia and it has been displacing the closely related F. culmorum in northern Europe. The 3ADON chemotype of F. graminearum is dominant in most northern areas, while the 15ADON chemotype of F. graminearum is predominating in Central and southern Europe. We suggest that the northern population of F. graminearum may be more specialized to oats than the southern population. Only low levels of F. culmorum DNA were found in a few oat samples and no correlation was found between F. culmorum DNA and DON levels. DNA levels of F. graminearum were in all cases in agreement with DON levels in 2011 and 2012, when DON was measured by gas chromatography-mass spectrometry (GC-MS). When the RIDA®QUICK SCAN kit results (DON) were compared to DNA levels of F. graminearum, the variation was much higher. The homogenization of the oats flour by grinding oats with 1 mm sieve seems to be connected to this variation. There was a significant correlation between the combined T-2 and HT-2 and the combined DNA levels of F. langsethiae and F. sporotrichioides in Finland in 2010-2012.