Infection Characteristics and Interaction of Three Species of Penicillium in the Postharvest Storage of ‘Gonggan’ Mandarin (Citrus reticulata)

Shaoping ZHANG　Chengxiang XU　Jinyan LAI　Yuchan PENG　Chunyan ZHONG　Minzhi LIANG

Abstract[Objectives] This study was conducted to understand the infection characteristics of Penicillium on ‘Gonggan’ mandarin （Citrus reticulata） and its effects on postharvest storage quality. [Methods]Nine inoculation treatments were designed and tested for the three identified Penicillium species， namely： three single inoculation experiments， four compound inoculation experiments， and two controls without inoculation. After treatment， the fruit was put into plastic film fresh-keeping bags and stored at a natural room temperature. The determined and analyzed indexes included fruit disease incidence， disease index， disease symptoms， disease spot expansion speed and weight loss rate， soluble solid content and organic acid content in different inoculation treatments. [Results] All ‘Gonggan’ fruit inoculated with Penicillium were diseased 2 d after inoculation， and completely rotted 14 d later. Of the three Penicillium species isolated and identified， Penicillium digitorum and its combinations were the most harmful to ‘Gonggan’ fruit. The hazard of Penicillium italicum was equal to or even weaker than that of Penicillium expansum. Eventually， P. digitorum dominated when coexisting on the same fruit. Different species of Penicillium obviously showed competition and antagonism in the process of growth and infection， especially for P. digitorum which was stronger， but other two species of Penicillium also had obvious self-protection ability. Low-temperature storage played a crucial role in the preservation of ‘Gonggan’ fruit， because it could significantly inhibit Penicillium infestation， and suppress fruit weight loss and the reduction of the contents of soluble solids and organic acids. [Conclusions]The infection characteristics and interaction relationship of Penicillium in the postharvest storage of ‘Gonggan’ fruit were revealed.

Key words‘Gonggan’ mandarin; Penicillium; Stab inoculation; Incidence characteristics; Fruit quality

‘Gonggan’ mandarin （Citrus reticulata） is a famous specialty in the Xijiang River Basin of Guangdong Province. The fruit has an average single fruit weight of 125 g， and is regularly shaped， golden in color， and the flesh tastes juicy， tender， smooth， refreshing and slagless， sweet and less sour， and is rich in fragrant. It has no seeds or few seeds. It concentrates the dual advantages of Citrus sinensis and Citrus reticulata， i.e.， the beautiful appearance of C. sinensis and the tender flesh and easy peeling of C. reticulata. ‘Gonggan’ is rich in nutrients and integrates the advantages of C. sinensis， sugar oranges and honey pomelos. It is known as the "King of Chinese Oranges"， and has successively won the honorary titles of "Top Ten Best Fruits in Lingnan" and "Chinese Famous Brand Fruit"[1]. The origin and main cultivation areas of ‘Gonggan’mandarin are currently limited to Zhaoqing City in the Xijiang River Basin in Guangdong with a recorded cultivation history more than 800 years， but the relatively professional research time is very short， and many fields are still blank[2].

The main diseases of citrus fruits during postharvest storage are blue mold and green mold. The pathogens are Penicillium italicum Wehmer and Penicillium digitatum Sacc， respectively.  In addition， there pathogens may also include Penicillium expansum， Oospora citriaurantii ex Persoon and Diplodia natalensis Evans[3-4]. The diseases in the post-harvest storage of ‘Gonggan’mandarin that we have identified are mainly blue mold and green mold， but it is felt that green mold infection is faster and more serious when it occurs， and the two diseases are intertwined and occur together， and it is difficult to distinguish them in many cases[5]. So far， no research has been reported on the pathogens and their infection characteristics in the post-harvest storage of ‘Gonggan’ fruit. In view of this， in this study， the infection characteristics and interaction of blue mold in the postharvest storage of ‘Gonggan’ fruit were studied.

Materials and Methods

Experimental materials

In mid-December， 2014， the ‘Gonggan’ fruit from 15 orchards in the Xijiang River Basin in Guangdong were collected， brought back to the laboratory and stored at room temperature and low temperature， respectively. From 86 diseased fruits stored for 30 d， after isolation， purification， morphological and PCR identification， the pathogens were P. italicum and P. digitatum mainly， and P. expansum was the second. Another Penicillium species was yet to be further identified. All strains were stored in a low-temperature refrigerator. The experiment was carried out in mid-December， 2015. Specially， the central part of the pericarp of ‘Gonggan’ fruit was gently drawn with a dissecting needle to form a linear wound， and then inoculated with the spores of the identified and confirmed three Penicillium species as pathogen materials （Fig. 1）， respectively.

Fruit treatment method

A total of 9 treatments were designed in the experiment： ① CK1： soaking only in 0.1% NaClO solution for 2 min， ② CK2： soaking in 0.1% NaClO solution for 2 min （the same below）， scratching the pericarp （all three lines）， free of inoculation with Penicillium， ③ inoculating only P. digitorum： +P. digitorum， ④ inoculating only P. expansum： +P. expansum， ⑤ only inoculating P. italicum： +P. italicum， ⑥ inoculating both P. digitorum and P. expansum： +P. digitorum+P. expansum， ⑦ inoculating both P. expansum and P. italicum： +P. expansum+P. italicum， ⑧ inoculating both P. digitorum and P. italicum： +P. digitorum+P. italicum， ⑨ inoculating P. digitorum， P. expansum and P. italicum. Fifteen pieces of fruit were treated for each treatment， in 3 replicates.

The tested Penicillium strains were cultured on PDA medium for 10 d （25-26 ℃） to obtain the tested spores， and the concentration of the spore suspension was 100 CFU/μl. When inoculating， ‘Gonggan’ fruit with no damage， no disease， uniform size， consistent maturity and intact stem was selected. Before inoculation， the ‘Gonggan’ fruit was first soaked in 0.1% sodium hypochlorite （NaClO） solution for 2 min， then taken out and placed on an ultra-clean bench to dry naturally. During inoculation， First， a dissecting needle was used to make equidistant cuts at 3 places， i.e.， above the equator line of the fruit （the inoculation site of P. expansum）， at the equatorial line （the inoculation site of P. digitorum）， and below the equator line （the inoculation site of P. italicum）. On the surface of ‘Gonggan’ fruit， the linear wounds were about 1.0 mm in depth and width and 10 mm in length. Then， corresponding freshly prepared Penicillium spore suspension （10 μl） was applied to the linear wound with a pipette， and after 2 h （the suspension had been air-dried）， the fruit was put into PE fresh-keeping bags according to the treatment and replicate. The bags were marked and stored， and the pathogens were cultured and observed at natural room temperature （16-18 ℃）.

Analysis methods

Spore suspension preparation and concentration calculation： On the surface of colonies on the PDA medium， 20 loops were gently scraped， added in sterile water and shaken well， and the concentration of the spore suspension （100 spores/μl） were calculated with a hemocytometer. Observation and measurement were performed daily （every 24 h） to record of disease incidence and disease spot growth after inoculation to the fruit， including the appearance time and symptoms of disease spots， and the incidence rates and disease indexes of the fruit diseases in different treatments were calculated for corresponding days. Since the disease condition of all the fruit inoculated with Penicillium had no significant difference after the 10 d， and all of them were completely rotten after the 14 d， the data of the first 10 d were used in the statistical analysis.

The incidence of fruit disease was calculated according to Incidence of fruit disease = Number of fruit diseased/Number of fruit involved in corresponding treatment （15）. Fruit disease index was calculated according to following formula： Disease index=[∑（Number of rotten fruit of each grade×Representative value of each grade）/（Total number of fruit in corresponding treatment×Representative value of the highest grade （7））]×100. The disease index was based on the degree of rot of the fruit after inoculation——the percentage （%） of the rotten area on the surface of the fruit， and was divided into 8 grades： grade 0： 0， grade 1： 0-1%， grade 2： 1%-5%， grade 3： 5%-10%; grade 4： 10%-20%， grade 5： 20%-50%， grade 6： 50%-80%， and grade 7： over 80%.

At 0， 4 and 8 d after inoculation， the weight loss rate and soluble sugar and organic acid contents of each treatment were tested and analyzed. Determination of fruit weight loss rate： On the day of storage， each bag of fruit was numbered and marked， and the weight （fresh weight） was measured. After storage， the fruit was measured regularly， and the weight loss rate was expressed as the percentage of the weight reduction of each marked fruit to its weight on the day of storage. The weight loss rate was calculated according to following formula： Weight loss rate （%）=（Weight at the time of storage-Weight after storage）/Weight at the time of storage×100.

Determination of fruit rot rate： The fruit with a disease spot equal to or greater than 0.50 cm in diameter or rotten fruit was recorded as rotten fruit. After storage， the number of rotten fruits in each bag was regularly counted according to the interval of days， and the rot rate was the percentage of the number of rotten fruits in the bag to the total number of stored fruit in the bag. The fruit rot rate was calculated according to following formula： Fruit rot rate （%） = Number of rotten fruit/Number of stored fruit × 100.

Determination of total soluble sugar， organic acid and vitamin C in fruit： With juice sacs as samples， the three indexes were determined according to NY/T2742-2015 Determination of soluble sugar in fruits and derived products—3，5-dinitrosalicylic acid colorimetry， GB/T12293-1990 Fruit and vegetable products—determination of titratable acidity （NaOH titration method） and GB5009.86-2016 Determination of ascorbic acid content in foods—2，6-dichlorophenol-indophenol method.

Statistical analysis of data

The experimental results were calculated for means and standard deviations and plotted in Mocrosoft Office Excel 2013 software， and the significance of differences between the mean values of different treatments was analyzed by Duncan's new multiple range method.

Results and Analysis

Differences in the incidence rate of ‘Gonggan’ fruit among different inoculation treatments

The daily observation results after inoculation showed that no disease occurred in the first 7 d of storage at room temperature for the two controls， and disease fruit was found in the CK without scratching of the pericarp on the 10 d and the incidence rate was only 3.5%; and the CK in which the pericarp was scratched began to appear diseased fruit on the 8 d of storage and the incidence rate was as high as 26.5%， and it was as high as 50.0% on the 10 d after inoculation and reached 100.0% on the 16 d. All ‘Gonggan’ fruit inoculated with Penicillium had no diseased fruit on the 1 d after inoculation， and diseased fruit appeared on the 2 d， and then the disease progressed very fast. The incidence rate was higher than 50% on the 5 d， higher than 70% on the 8 d， and 90.0%-100% on the 10 d， but the differences between different treatments were still relatively large. Table 1 shows the differences in the incidence rate of ‘Gonggan’ fruit on the 2， 4， 6， 8 and 10 d after inoculation with single Penicillium alone or two or three in combination. As can be seen from Table 1， ‘Gonggan’ fruit that was inoculated with one of P. digitorum， P. expansum and P. italicum alone had significantly lower incidence rates than the fruit inoculated with them in combination in the first 8 d after inoculation. For the infectivity of the three Penicillium species， P. digitatum was the strongest， P. italicum and P. expansum had the same infectivity， but P. italicum was stronger. Among the 4 kinds of compound inoculation treatments， the fruit inoculated with P. digitorum and P. italicum simultaneously had the highest incidence rate， and showed a similar regularity to that of single inoculation.

Differences in the disease index of ‘Gonggan’ fruit among different inoculation treatments

Disease index is another important index that reflects the severity of a disease. With the same incidence rate， the disease index can be very different. Therefore， disease index can reflect the severity of a disease in a more detailed and accurate manner. The response trends of disease index and incidence rate of ‘Gonggan’ fruit after inoculation with Penicillium were similar， and they both increased rapidly with the increase of storage days after inoculation， and both were significantly different from the two controls， but the differences between different inoculation treatments were greater. From the analysis results in Table 2， it could be seen that when the three species of Penicillium were inoculated individually， the disease index was the highest with the inoculation of P. digitatum， followed by the inoculation of P. expansum， and the treatment inoculated with P. italicum showed the smallest disease index， further indicating that their infectivity was significantly different. When the two main pathogenic Penicillium species， P. digitorum and P. italicum， which are generally considered to be the main pathogens， were inoculated with P. expansum， respectively， the disease index of the combination of P. italicum and P. expansum was always significantly greater than that of P. digitatum and P. italicum.  Simultaneously inoculated with P. digitatum and P. italicum， the ‘Gonggan’ fruit showed a disease index in the early stage comparable to that inoculated with P. italicum and P. expansum， but significantly greater than that inoculated with P. digitatum and P. expansum， and in the later stage， the disease index was significantly smaller than that of the combination of P. italicum and P. expansum， and significantly larger than that of the combination of P. digitorum and P. expansum. Simultaneously inoculated with the three species of Penicillium， the ‘Gonggan’ fruit showed the smallest disease index during the 10-day period， and the disease was relatively light （Table 2）.  These results clearly showed that the species of pathogens were closely related to the storage condition of ‘Gonggan’ fruit; the pathogenicity of P. italicum was significantly weaker than that of P. digitorum， and P. digitorum should be the most important Penicillium to be controlled during the storage period of ‘Gonggan’ fruit; and the three main pathogens had significant competition and antagonism in different combinations during the infection process.

Differences in disease symptoms and lesion expansion speed of ‘Gonggan’ fruit after inoculation

On the 2 d after inoculation， disease spots first grew on the ‘Gonggan’ fruit inoculated with P. expansum， P. digitorum and P. digitorum+P. expansum+P. italicum. The symptoms of each treatment in the initial disease stage were similar. First， white colonies grew on the wound， with irregular edges， and the diseased part was soft rot and hygrophanous.

In the fruits inoculated with a single strain （Fig. 2）， mycelia first appeared on P. expansum and P. italicum， but P. digitatum grew the fastest and had the largest colony volume， followed by P. expansum and P. italicum. After 3 d of inoculation， in colonies of P. expansum and P. italicum， a cyan powdery mold layer was present with a narrow white ring around the edge， while green mold colonies were powdery green in appearance with a thick white ring rounding the outer edge. After that， Penicillium colonies all grew rapidly， and the conditions of all the fruit inoculated with Penicillium had no significant differences by 10 d of storage. On the 14 d， all the fruit was completely covered by colonies， and a large amount of mucus was exuded， which made it difficult to maintain the original shape of the fruit.

The three species of Penicillium clearly competed with each other during growth （Fig. 3）.  With the continuous growth of mold mycelia， especially the rapid growth of P. digitatum， a clear demarcation area was formed between colonies， and P. digitatum surrounds the slower-growing P. italicum and P. expansum， but form obvious and relatively regular edges near each other， which clearly showed that there was a certain antagonism between them. Therefore， even the fast-growing Penicillium did not spread to the diseased part caused by other Penicillium at the beginning， but expanded and spread to other parts that had not been infected， which alleviated the disease instead.

Differences in important quality indexes of ‘Gonggan’ fruit among different inoculation treatments

With the increase of storage days， the weight loss rate of ‘Gonggan’ fruit increased， the contents of soluble solids and organic acids decreased， but the fruit free of Penicillium inoculation was significantly different. It can be seen from Table 3 that CK had the smallest change， followed by CK. Meanwhile， the content of soluble solids showed a slight increase trend， and the two indexes of weight loss rate and soluble solid content still showed significant differences between the two treatments， that is， CK was significantly greater than CK. It can also be seen from Table 3 that after inoculation with the three Penicillium species， regardless of combinations， the fruit weight loss rate of ‘Gonggan’ fruit was significantly greater than that of the two controls， and the soluble solid and organic acid contents were significantly lower than those of the two controls， but there were still major differences among different inoculation treatments， especially in terms of weight loss rate and soluble solid content. In general， all combinations including P. digitorum showed higher fruit weight loss rates， and the contents of soluble solids and organic acids was lower; and single or combined inoculation of P. italicum and P. expansum had similar effects on the three key quality indexes of ‘Gonggan’ fruit， which were similar to the previous responses of the incidence rate and disease index.

Discussion

Basic identification of the main pathogens in postharvest storage is an important technological progress in postharvest biological research and fresh-keeping practice of ‘Gonggan’ mandarin. However， the specific infection characteristics and interaction relationship were not clear before.  From the experimental results of this study， it could be seen that the disease incidence， disease index， disease symptoms， disease spot expansion speed， weight loss rate， soluble solid content， organic acid content and other important quality indexes of ‘Gonggan’ fruit differed significantly with different inoculation treatments. In general， P. digitatum and all its combinations expanded rapidly and had the most serious damage; P. italicum had an early onset， but grew slowly， and its hazard was equal to or even weaker than that of P. expansum; and an important finding was that P. digitorum （green mildew） eventually dominated when co-existing on the same fruit. In people's common knowledge and management， it is generally said that citrus storage diseases are mainly blue mold and green mold， but in fact green mold is much more harmful than blue mold. In the practice of postharvest storage of ‘Gonggan’ mandarin， how to prevent and control P. digitorum and the green mold disease caused by it should be the key.

The simultaneous infection of multiple pathogens in fruit storage usually aggravates and accelerates the disease. However， the inoculation treatment of the three species of Penicillium in this study found that compared with single inoculation， whether two or three species of Penicillium were inoculated simultaneously， the disease of ‘Gonggan’ fruit was not significantly aggravated. On the contrary， different species of Penicillium competed and antagonized in the process of growing and infecting， especially P. digitorum， but other two species of Penicillium also apparently had the ability to protect the "territory" that had been formed. It might be mainly due to "allelopathy"， but what the specific secondary metabolites are is currently unknown. It is an interesting and useful phenomenon. At present， the use of yeast to control blue mold and green mold in citrus has been reported. Geng et al.[6] screened and identified an antagonistic yeast， Kluyueromyces marxianus， which can effectively control postharvest green mold of citrus from different fruits. Luo et al.[7] isolated an antagonistic yeast， K. marxianus， from the surface of papaya. Inoculation with the antagonistic fungus and pathogens could induce the increase of PPO， POD and PAL activity in citrus fruit， and the antimicrobial mechanism of the antagonistic yeast against citrus green mold was related to the competition for nutrients and space and the induction of resistance-related enzymes[7]. "Controlling microbes with microbes"， "suppressing microbes with microbes" and biological control can all be used for reference and utilization in the practice of postharvest storage and preservation of ‘Gonggan’ fruit.

We have found in the previous study that during the 90-day storage， the contents of vitamin C and organic acids in refrigerated ‘Gonggan’ fruit was significantly lower than that of the control （stored at room temperature）; the content of soluble sugar changed little， and the ratio of sugar to acid in the fruit had no significant difference from that before storage; and the accumulation of important odor substances ethanol and acetaldehyde in the fruit was significantly inhibited， and the daily average weight loss rate， malondialdehyde content and relative permeability of the cytoplasmic membrane in the fruit were significantly lower than those of the control. Electron microscope observation showed that the wax content of the pericarp of ‘Gonggan’ fruit stored at room temperature decreased rapidly， and refrigeration effectively protected the pericarp wax coat， and the micromorphological structure of the pericarp surface remained well， and was significantly different from the control. Chromatography-mass spectrometry analysis showed that refrigerated storage significantly inhibited the reduction of the wax content in the pericarp of ‘Gonggan’ fruit， and the total wax and the content of the four main long-chain components were significantly higher than those of the control， and the branched alkanes， alkenes and olefine aldehydes in the pericarp wax were still significant at 90 d of storage[8-10]. Obviously， the good fresh-keeping effect of cold storage is closely related to the effective protection of the wax content， chemical composition and surface micro-morphological structure of fruit pericarps. It can be clearly seen from the experimental results in this study that low-temperature storage played a crucial role in the preservation of ‘Gonggan’ fruit， because it could significantly inhibit Penicillium infestation， and suppress fruit weight loss and the reduction of the contents of soluble solids and organic acids.

Conclusions

Of the three Penicillium species isolated and identified， P. digitorum and its combinations were the most harmful to ‘Gonggan’ fruit. The hazard of P. italicum was equal to or even weaker than that of P. expansum. Eventually， P. digitorum dominated when coexisting on the same fruit. Different species of Penicillium obviously showed competition and antagonism in the process of growth and infection， especially for P. digitorum which was stronger， but other two species of Penicillium also had obvious self-protection ability. Low-temperature storage played a crucial role in the preservation of ‘Gonggan’ fruit， because it could significantlyinhibit Penicillium infestation， and suppress fruit weight loss and the reduction of the contents of soluble solids and organic acids.

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